Dengue virus e-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes

ABSTRACT

Described herein are dengue virus E-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes associated with immune enhancement. The disclosed dengue virus E-glycoproteins optionally further include mutations that introduce a strong CD4 T cell epitope. The disclosed E-glycoprotein polypeptides, or nucleic acid molecules encoding the polypeptides, can be used, for example, in monovalent or tetravalent vaccines against dengue virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/549,348, filed Oct. 20, 2011, which is herein incorporated byreference in its entirety.

FIELD

This disclosure concerns immunogenic dengue virus compositions in whichimmunodominant cross-reactive epitopes have been removed, and their usefor redirecting an immune response for safe and efficacious denguevaccination.

BACKGROUND

Dengue viruses (DENV) are the most prevalent arthropod-borne viralpathogens infecting humans. These mosquito-transmitted viruses, membersof the Flaviviridae, are endemic to most tropical and sub-tropicalcountries with nearly half of the world's population living at risk ofDENV infection and resulting in over a million estimated infectionsannually (Mackenzie et al., Nat Med 10:S98-109, 2004; Gubler, Arch MedRes 33:330-342, 2002). Infection with DENV can cause a broad range ofsymptoms, ranging from subclinical, to the self-limiting flu-likeillness dengue fever (DF), to the more severe and life-threateningdengue hemorrhagic fever and shock syndrome (DHF/DSS) characterized byincreased vascular permeability producing plasma leakage, severethrombocytopenia and hypotension leading to circulatory collapse(Gubler, Novartis Found Symp 277:3-16; discussion 16-22, 71-13, 251-253,2006). DENV prevalence, infection rates, and disease severity haveincreased exponentially since the middle of the last century (Guzman etal., Nat Rev Microbiol 8:S7-S16, 2010). Despite decades of interest,need, and effort there remains no available dengue vaccine and vaccinecandidates continue to run into roadblocks and safety concerns both inpre-clinical development and in clinical trials (Guy et al., Hum Vaccin6(9), Epub Sep. 16, 2010; Miller, Curr Opin Mol Ther 12:31-38, 2010;Thomas, J Infect Dis 203:299-303, 2011; Murphy and Whitehead, Annu RevImmunol 29:587-619, 2011).

Dengue vaccine development is plagued by a number of biological andimmunological challenges that also affect vaccinology for othermulti-strain pathogens. These include the necessity for a tetravalentvaccine inducing balanced immunity, the lack of an animal model for DENVdisease, and concerns regarding vaccine-induced severe DENV pathology(Murphy and Whitehead, Annu Rev Immunol 29:587-619, 2011; Morens andFauci, JAMA 299:214-216, 2008). DENV immune responses are bothprotective and pathogenic and it is this duality that directly impedesvaccine development (Rothman, J Clin Invest 113:946-951, 2004). Thereare four closely related yet phylogenetically distinct DENV serotypes(DENV-1, -2, -3, and -4) and infection with any one serotype appears toinduce life-long serotype-specific immunity, yet cross-protectionbetween serotypes is limited and transient (Sabin, Am J Trop Med Hyg1:30-50, 1952; Kuno, Adv Virus Res 61:3-65, 2033). Thus, in endemicregions individuals are susceptible to up to four different DENVinfections.

Although there are a number of risk factors associated with DHF such asvirus and host genetics, by far the strongest risk factor for severedengue pathology is secondary infection with a previously unencountered(heterologous) serotype (Murphy and Whitehead, Annu Rev Immunol29:587-619, 2011). This association explains the exponential increase ofDHF/DSS in recent decades as co-circulation and simultaneoustransmission of the four DENV serotypes increases both temporally andgeographically (Mackenzie et al., Nat Med 10:S98-109, 2004; Gubler,Novartis Found Symp 277:3-16; Guzman et al., Nat Rev Microbiol 8:S7-S16,2010). In humans, increasing viral load correlates with DENV diseaseseverity and a large body of evidence points to the importance of immuneenhancement being a causal factor for the increased viral loadsassociated with DHF (Vaughn et al., J Infect Dis 181:2-9, 2000; Libratyet al., J Infect Dis 185:1213-1221, 2002).

SUMMARY

Disclosed herein are dengue virus E-glycoprotein polypeptides containingmutations that eliminate immunodominant cross-reactive epitopesassociated with immune enhancement. In some cases, the dengue virusE-glycoproteins further include mutations that introduce a strong CD4 Tcell epitope. The disclosed E-glycoprotein polypeptides, or nucleic acidmolecules encoding the polypeptides, can be used, for example, inmonovalent or tetravalent vaccines against dengue virus.

Provided herein are cross-reactivity reduced dengue virus E-glycoproteinpolypeptides having amino acid substitutions at residues correspondingto positions 106, 107, 310 and 311, and either position 364 or position389 of dengue serotype 1 (DENV-1) E-glycoprotein (SEQ ID NO: 1). Theprovided E-glycoprotein polypeptides optionally further includemutations corresponding to positions 468, 478, 482 and 487 of DENV-1E-glycoprotein (SEQ ID NO: 1).

In some embodiments, the cross-reactivity reduced dengue virusE-glycoprotein polypeptide is selected from: (1) a dengue serotype 1virus (DENV-1) E-glycoprotein polypeptide comprising an arginine atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a lysine at position 311, a glutamine at position 364, anisoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO: 1; (2) a dengue serotype 2 virus (DENV-2) E-glycoproteinpolypeptide comprising an arginine at position 106, an aspartic acid atposition 107, an glutamic acid at position 310, an arginine at position311, an arginine at position 364, an isoleucine at position 468, athreonine at position 478, a valine at position 482 and a leucine atposition 487, numbered with reference to SEQ ID NO: 2; (3) a dengueserotype 3 virus (DENV-3) E-glycoprotein polypeptide comprising anaspartic acid at position 106, an aspartic acid at position 107, anaspartic acid at position 308, a lysine at position 309, an asparticacid at position 362, an isoleucine at position 466, a threonine atposition 476, a valine at position 480 and a leucine at position 485,numbered with reference to SEQ ID NO: 3; and (4) a dengue serotype 4virus (DENV-4) E-glycoprotein polypeptide comprising a glutamic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a glutamine at position 311, a glutamine at position 389,an isoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO: 4.

Isolated virus-like particles (VLPs) comprising the disclosedpolypeptides, recombinant nucleic acid molecules encoding the disclosedpolypeptides and VLPs, vectors comprising the recombinant nucleic acidmolecules and isolated cells comprising the vectors are further providedby the present disclosure.

Also provided are compositions comprising the cross-reactivity reducedE-glycoprotein polypeptides, VLPs, recombinant nucleic acid moleculesand vectors disclosed herein.

Further provided are methods of eliciting an immune response in asubject against dengue virus by administering to the subject atherapeutically effective amount of the cross-reactivity reducedE-glycoprotein polypeptides, VLPs, recombinant nucleic acid molecules,vectors and compositions disclosed herein. In some examples, anE-glycoprotein polypeptide (or recombinant nucleic acid moleculeencoding the polypeptide) from a single dengue virus serotype isadministered as a monovalent vaccine. In other examples, anE-glycoprotein polypeptide (or recombinant nucleic acid moleculeencoding the polypeptide) from each of the four dengue virus serotypesis administered as a tetravalent vaccine. The disclosed nucleic acidmolecules encoding the cross-reactivity reduced dengue virusE-glycoproteins can be used, for example, in DNA prime-protein boostvaccine strategies.

The foregoing and other features, and advantages of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: Structural locations of the pVD1-CRR envelope (E) proteincross-reactive epitope knock out substitutions. The locations of theDENV-1 E substitutions introduced to construct pVD1-CRR are mapped onthe crystal structure of the homologous DENV-2 E protein dimer (Modis etal., Proc Natl Acad Sci USA 100:6986-6991, 2003). (FIG. 1A) Locations ofthe pVD1-CRR substitutions (spheres) on a ribbon diagram of the mature Edimer as they appear looking straight down toward the virion surface.The three structural domains are labeled (EDI, EDII and EDIII). Thefusion peptide is also labeled. The glycans in EDI (N67) and EDII (N153)are depicted as ball and stick representations. (FIG. 1B) Side-view ofthe E protein dimer. (FIG. 1C) Close-up of the EDII_(FP) and EDIIIregions of one monomer of the E dimer as it appears in panel (B). Theside chains of two residues in the EDII_(FP) and three residues in EDIIIwhere CRR substitutions were introduced are depicted as ball and stickrepresentations and labeled with the introduced substitutions. The twoantigenic regions EDII_(FP) and EDIII_(CR) are noted with circlesroughly representing the size of an IgG footprint binding to theseregions.

FIGS. 2A-2F: Protective Efficacy—Both pVD1-WT and pVD1-CRR vaccinesprotect AG129 mice against lethal DENV-1 challenge. (FIGS. 2A-2D) 12week post vaccination immunogenicity of AG129 mice immunized (i.m.) with100 μg of pVD1-WT or pVD1-CRR vaccines at 0, 4 and 8 weeks. Geometricmeans and 95% confidence intervals are depicted unless otherwise noted.All end-point titers were log₁₀ transformed and statistical significancewas determined using the Mann-Whitney test to account for non-normalityof some transformed data. (FIG. 2B) and (FIG. 2C) both used a one-tailedtest since there was an a priori expectation that pVD1-CRR immunizedmice would exhibit reduced EDII_(FP) recognizing IgG. (FIG. 2A) DENV-1total IgG end-point titers 12 weeks post immunization. (FIG. 2B) Percentof total DENV-1 IgG recognizing immunodominant EDII_(FP) epitopes,arithmetic means and 95% CI depicted. (FIG. 2C) Calculated DENV-1EDII_(FP) IgG end-point titers. (FIG. 2D) DENV-1 50% antigenfocus-reduction micro neutralization titers (FRμINT₅₀). A t-test wasutilized here as both data sets were normally distributed. (FIG. 2E)pVD1-WT and pVD1-CRR immunized AG129 mice were challenged with 1.1×10⁵FFU of DENV-1 (Mochizuki strain, i.p.) at 12 weeks. Kaplan-Meiersurvival curves are shown. Both pVD1-WT (n=18) and pVD1-CRR (n=18)vaccinated animals had the same survival (94%), which was highlysignificant in comparison to naïve mice (n=4, p=0.0003). (FIG. 2F) Fourmice from each vaccine treatment (not included in the survival curves)were a priori scheduled to be euthanized 3 and 8 DPC. DENV-1 titers(FFU/mL) were log₁₀ transformed and analyzed using 2-way ANOVA; errorbars represent standard error of the mean (SEM). Vaccine treatment washighly significant (p<0.0001). Bonferroni post-tests indicated thatviremia of each vaccinated group was significantly lower than for naïvemice (n=2) 3 DPC (p<0.001), only pVD1-CRR vaccinated mouse viremia wassignificantly lower than naïve mice (n=2) 8 DPC (p<0.01) and there wasno difference between WT or CRR vaccinated mouse viremia 3 or 8 DPC.

FIGS. 3A-3F: DENV-1 CRR vaccine stimulates reduced levels ofimmunodominant cross-reactive EDII_(FP) IgG and reduces enhanced DENV-2mortality. (FIGS. 3A-C) 12 week post vaccination immunogenicity of AG129mice immunized (i.m.) with 100 μg of pVD1-WT or pVD1-CRR vaccines.Geometric means and 95% confidence intervals are depicted. End-pointtiters were log₁₀ transformed and statistical significance wasdetermined using the two-tailed Mann-Whitney test. (FIG. 3B) and (FIG.3C) both used a one-tailed test since there was an a priori expectationthat pVD1-CRR immunized mice would exhibit reduced EDII_(FP) recognizingIgG. (FIG. 3A) DENV-1 total IgG end-point titers 12 weeks postimmunization. (FIG. 3B) Percent of total DENV-1 IgG recognizingimmunodominant EDII_(FP) epitopes, arithmetic means and 95% CI depicted.(FIG. 3C) Calculated DENV-1 EDII_(FP) IgG end-point titers. (FIG. 3D)Survival of pVD1-WT and pVD1-CRR immunized AG129 mice followingsublethal heterologous DENV-2 challenge. 12 weeks following vaccination,immunized mice (n=22) or age matched naïve controls (n=8) werechallenged (i.p.) with 4.2×10⁵ FFU of DENV-2 S221. Kaplan-Meier survivalcurves and p values are shown. All naïve mice survived virus challengewith minimal signs of morbidity, pVD1-WT vaccinated mice suffered 95%mortality from enhanced DENV-2 disease (p<0.0001, compared to naive).pVD1-CRR immunized mice exhibited 68% survival which did not differ fromnaïve mouse survival (100%, p=0.0769) yet was significantly greater thanpVD1-WT immunized mouse survival (4.5%, p<0.0001). The Bonferronimultiple comparison adjusted a=0.017. (FIG. 3E) pVD1-CRR vaccinated miceexhibited a rapid, large magnitude increase in DENV-2 neutralizingantibody titers following DENV-2 challenge. FRμNT₅₀ titers determinedwith DENV-2 16681 virus on Vero cells, log₁₀ transformed and thetransformed data analyzed by 2-way ANOVA (p<0.0001). Bonferroni posttestsignificance is depicted with asterisks, error bars represent SEM. 0, 3,4, and 5 DPC n=4, 4, 5 and 9 for WT and 4, 4, 1, and 3 for CRR immunizedmice, respectively. (FIG. 3F) Viremia of pVD1-WT vaccinated miceincreased rapidly 3-5 days after DENV-2 S221 challenge, whereas pVD1-CRRvaccinated mice had 30-fold, 40-fold, and at least 1800-fold lowerviremia 3, 4, and 5 DPC (all CRR mice<10 FFU/mL) than did WT immunizedmice. Virus titers were log₁₀ transformed and the transformed dataanalyzed by 2-way ANOVA (p<0.0001). Bonferroni posttest significance isdepicted with asterisks, error bars represent SEM. 3, 4 and 5 DPC n=4,5, and 6 for WT and 4, 1, and 3 for CRR immunized mice, respectively.

FIGS. 4A-4C: Pathophysiology of enhanced DENV disease in vaccinatedAG129 mice. (FIG. 4A) Enhanced DENV disease associated vascular leakpathology of pVD1-WT and -CRR vaccinated mice following sub-lethalheterologous DENV-2 infection. No pathology was visible in naïve orvaccinated mice 3 DPC, yet by 4 and 5 DPC mice succumbing to enhanceddisease exhibited severe vascular leakage associated pathology. The samemouse is shown in the bottom two photos to highlight the severeintestinal hemorrhage observed in pVD1-WT but not in pVD1-CRR vaccinatedmice (arrows). (FIG. 4B) Histology of uninfected, pVD1-WT, and pVD1-CRRvaccinated mouse livers 3 DPC. The top row is hematoxylin and eosinstained liver sections and the bottom row is immunohistochemistry forDENV-2 NS 1 protein of the same individual animals. NS1+ mononuclearinflammatory cells and sinusoidal endothelial cells stain positive intheir cytoplasm and are visible (arrows) in liver tissue from vaccinatedmice but not uninfected mice. Multiple sections from multiple animalswere examined and single representatives are shown. All photos weretaken at 400× magnification. (FIG. 4C) In vitro DENV-2 enhancement bypVD1-WT and pVD1-CRR vaccinated serum 12 weeks following vaccinationsand one day prior to DENV-2 challenge. Consistent with the in vivoresults, pVD1-WT vaccinated serum significantly enhanced DENV-2infection whereas pVD1-CRR vaccinated serum did not (p=0.0012). The dataare representative of two independent experiments of 4 pools of 6-7individual serum specimens for each vaccine treatment and were analyzedwith 2-way ANOVA and Bonferroni post test significance at individualdilutions is depicted with asterisks.

FIGS. 5A-5F: DENV-1 CRR vaccination sculpts immunity to redirectsecondary immunity to heterologous dengue infection. AG129 mice wereimmunized (i.m.) with 100 μg of pVD1-WT or pVD1-CRR vaccines andchallenged at 12 weeks with heterologous DENV-2 S221 (4.2×10⁵ FFU,i.p.). Arithmetic means and SEM are depicted in A, B, D, and E andanalyzed with two-way ANOVA. Bonferroni post test significance isdepicted with asterisks. (FIG. 5A) DENV-1 total IgG end-point titers ofpVD1-WT and -CRR immunized mice 3, 4, and 5 DPC with DENV-2. Three, 4,and 5 DPC n=4, 3, and 8 and 4, 1, and 3 for WT and CRR vaccines,respectively. (FIG. 5B) DENV-2 total IgG end-point titers of pVD1-WT and-CRR vaccinated mice. (FIG. 5C) Percent DENV-1 epitope-specificresponses pre- and post-challenge for IgG recognizing immunodominantcross-reactive EDII_(FP) epitopes. pVD1-WT immunized mice had largerpopulations of EDII_(FP) post DENV-2 challenge than did pVD1-CRRimmunized mice (ave=35.5% and 7.65% respectively, p=0.0004). pVD1-WTimmunized mice also exhibited an increase in EDII_(FP) IgG from pre- topost-DENV-2 challenge (ave=17.7% and 35.5% respectively, p=0.0057.Geometric means and 95% CI are depicted. Statistical significance wasdetermined with a one-tailed Mann-Whitney U. The Bonferroni a=0.025.(FIG. 5D, 5E) DENV-2 epitope specific IgG responses post DENV-2challenge of pVD1-WT and pVD1-CRR immunized AG129 mice; 3, 4, and 5 DPCn=4, 5, and 9 and 4, 1, and 2 for WT and CRR immunized mice,respectively. (FIG. 5D) Percent DENV-2 IgG recognizing immunodominantcross-reactive EDII_(FP) epitopes. (FIG. 5E) Percent DENV-2 IgGrecognizing E protein epitopes outside of immunodominant EDII_(FP) andEDIII_(CR) antigenic regions (Non-EDII_(FP)EDIII_(CR)). (FIG. 5F)FRμNT₅₀ titers of pVD1-WT and -CRR immunized mouse sera 3 DPC withDENV-2 against DENV-1, DENV-2, DENV-3 and DENV-4. Bars represent GMT,statistical significance was determined with a one-tailed t-test. Alltiters were log₁₀ transformed prior to analysis.

FIG. 6: DENV-1 CRR vaccination redirects immune responses to DENV-2 toincrease a diversity of neutralizing antibodies. Percent blocking oflabeled monoclonal antibodies (MAbs) by pVD1-WT and pVD1-CRR immunizedmice 3 DPC to DENV-2 as determined by blocking ELISA. AG129 mice wereimmunized (i.m.) with 100 μg of pVD1-WT or pVD1-CRR vaccines andchallenged at 12 weeks with a sub-lethal dose of heterologous DENV-2S221 (4.2×10⁵ FFU, i.p.). Three DPC sera of the four mice from eachvaccine treatment were pooled and percent blocking was determinedindependently four times in duplicate. To facilitate comparison betweenvaccine treatments across the different MAbs, x-fold increases inblocking by pVD1-CRR vaccinated sera relative to pVD1-WT vaccinated seraare depicted above the x axis for each MAb. Below the MAbs, serotypespecificity is depicted (DENV-2=DENV-2 type specific, subcomp=DENVsubcomplex cross-reactive, complex=DENV complex cross-reactive andcomplex+=supercomplex cross-reactive). Below specificity, p values froma two-tailed t-test comparing percent blocking for each vaccinated seraare depicted. Below that the presence or absence of the epitoperecognized by each MAb on the DENV-1 CRR VLP antigen is depicted (allMAbs recognize epitopes found in the DENV-2 challenge virus). Forexample, 3H5 and 9A3D-8 as DENV-2 specific antibodies are absent on bothpVD1-WT and pVD1-CRR VLP antigens; 9D12 and 1A1D-2 reactivity areablated by the EDIII_(CR) substitutions in the pVD1-CRR plasmid (Table2) but present in the WT DENV-1 antigen; and 10-D35A, D3-5C9-1, and 1B7reactivities were not altered by the substitutions introduced into thepVD1-CRR plasmid and hence are present on both DENV-1 WT and CRRimmunization antigens. FRμNT₅₀ titers for each MAb against DENV-2, -1,-3, and -4 in μg/mL are also shown.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Oct. 5, 2012, 67.3 KB, which is incorporated byreference herein. In the accompanying Sequence Listing:

SEQ ID NO: 1 is the amino acid sequence of a mutant DENV-1 E proteinwith reduced cross-reactivity. This sequence comprises 80% DENV-1 Eprotein sequence at the N-terminus and 20% Japanese encephalitis virus(JEV) sequence at the C-terminus.

SEQ ID NO: 2 is the amino acid sequence of a mutant DENV-2 E proteinwith reduced cross-reactivity.

SEQ ID NO: 3 is the amino acid sequence of a mutant DENV-3 E proteinwith reduced cross-reactivity. This sequence comprises 80% DENV-3 Eprotein sequence at the N-terminus and 20% Japanese encephalitis virus(JEV) sequence at the C-terminus.

SEQ ID NO: 4 is the amino acid sequence of a mutant DENV-4 E proteinwith reduced cross-reactivity. This sequence comprises 80% DENV-4 Eprotein sequence at the N-terminus and 20% Japanese encephalitis virus(JEV) sequence at the C-terminus.

SEQ ID NO: 5 is the amino acid sequence of the DENV-1 prM/E proteinencoded by the pVD1iRDDKQ TMD construct.

SEQ ID NO: 6 is the amino acid sequence of the DENV-2 prM/E proteinencoded by the pVD2iRDERR TMD construct.

SEQ ID NO: 7 is the amino acid sequence of the DENV-3 prM/E proteinencoded by the pVD3iDDDKD TMD construct.

SEQ ID NO: 8 is the amino acid sequence of the DENV-4 prM/E proteinencoded by the pVD4iEDDQQ TMD construct.

SEQ ID NO: 9 is the nucleotide sequence of the pVD1iRDDKQ TMD construct.

SEQ ID NO: 10 is the nucleotide sequence of the pVD2iRDERR TMDconstruct.

SEQ ID NO: 11 is the nucleotide sequence of the pVD3iDDDKD TMDconstruct.

SEQ ID NO: 12 is the nucleotide sequence of the pVD4iEDDQQ TMDconstruct.

SEQ ID NOs: 13-19 are the nucleotide sequences of mutagenic primers.

DETAILED DESCRIPTION I. Abbreviations

ADE antibody-dependent enhancement

CRR cross-reactivity reduced

DENV dengue virus

DF dengue fever

DHF dengue hemorrhagic fever

DPC days post challenge

DSS dengue shock syndrome

E envelope protein

ELISA enzyme-linked immunosorbent assay

ES-ELISA epitope-specific ELISA

FFU focus-forming unit

FRμNT focus-reduction micro neutralization assay

HRP horseradish peroxidase

IHC immunohistochemistry

i.m. intramuscular

i.p. intraperitoneal

JEV Japanese encephalitis virus

M membrane protein

MAb monoclonal antibody

MHIAF murine hyper-immune ascitic fluid

OD optical density

prM premembrane protein

TLAV tetravalent live attenuated virus

TMD transmembrane domain

VLP virus-like particle

VRP viral replicon particle

WNV West Nile virus

WT wild type

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in

Benjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance or vehicle that non-specifically enhances theimmune response to an antigen. Adjuvants can include a suspension ofminerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (for example, Freund's incomplete adjuvant),sometimes with the inclusion of killed mycobacteria

(Freund's complete adjuvant) to further enhance antigenicity.Immunostimulatory oligonucleotides (such as those including a CpG motif)can also be used as adjuvants (for example, see U.S. Pat. Nos.6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068;6,406,705; and 6,429,199). Adjuvants also include biological molecules,such as costimulatory molecules. Exemplary biological adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L and 41 BBL.

Administer: As used herein, administering a composition (e.g. animmunogenic composition) to a subject means to give, apply or bring thecomposition into contact with the subject. Administration can beaccomplished by any of a number of routes, such as, for example,topical, oral, subcutaneous, intramuscular, intraperitoneal,intravenous, intrathecal and intramuscular.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: An immunoglobulin molecule produced by B lymphoid cells with aspecific amino acid sequence. Antibodies are evoked in humans or otheranimals by a specific antigen (immunogen). Antibodies are characterizedby reacting specifically with the antigen in some demonstrable way,antibody and antigen each being defined in terms of the other.“Eliciting an antibody response” refers to the ability of an antigen orother molecule to induce the production of antibodies.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens.

Cross-reactivity reduced dengue virus E-glycoprotein: A dengue virusE-glycoprotein containing amino acid substitutions that result in theremoval of immunodominant epitopes associated with immune enhancement.

Dengue virus (DENV): An RNA virus of the family Flaviviridae, genusFlavivirus. The dengue virus genome encodes the three structuralproteins (C, prM and E) that form the virus particle and sevennon-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) that areonly found in infected host cells, but are required for replication ofthe virus. There are four serotypes of dengue virus, referred to asDENV-1, DENV-2, DENV-3 and DENV-4. All four serotypes can cause the fullspectrum of dengue disease. Infection with one serotype can producelifelong immunity to that serotype. However, severe complications canoccur upon subsequent infection by a different serotype. Dengue virus isprimarily transmitted by Aedes mosquitoes, particularly A. aegypti.Symptoms of dengue virus infection include fever, headache, muscle andjoint pain and a skin rash similar to measles. In a small percentage ofcases, the infection develops into a life-threatening dengue hemorrhagicfever, typically resulting in bleeding, low platelet levels and bloodplasma leakage, or into dengue shock syndrome characterized bydangerously low blood pressure.

Envelope (E) glycoprotein: A flavivirus structural protein that mediatesbinding of flavivirus virions to cellular receptors on host cells. Theflavivirus E protein is required for membrane fusion, and is the primaryantigen inducing protective immunity to flavivirus infection. FlavivirusE protein affects host range, tissue tropism and viral virulence. Theflavivirus E protein contains three structural and functional domains,DI-DIII. In mature virus particles the E protein forms head to tailhomodimers lying flat and forming a dense lattice on the viral surface.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigenic polypeptide or vaccine. An immune response can includeany cell of the body involved in a host defense response, including forexample, an epithelial cell that secretes an interferon or a cytokine.An immune response includes, but is not limited to, an innate immuneresponse or inflammation. As used herein, a protective immune responserefers to an immune response that protects a subject from infection(prevents infection or prevents the development of disease associatedwith infection). Methods of measuring immune responses are well known inthe art and include, for example, measuring proliferation and/oractivity of lymphocytes (such as B or T cells), secretion of cytokinesor chemokines, inflammation, antibody production and the like.

Immune stimulatory composition: A term used herein to mean a compositionuseful for stimulating or eliciting a specific immune response (orimmunogenic response) in a subject. The immune stimulatory compositioncan be a protein antigen or a nucleic acid molecule (such as vector)used to express a protein antigen. In some embodiments, the immunogenicresponse is protective or provides protective immunity, in that itenables the subject to better resist infection with or diseaseprogression from the flavivirus against which the immune stimulatorycomposition is directed.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of an immune stimulatory composition is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. Such a response may comprise, for instance, generation of anantibody specific to one or more of the epitopes provided in the immunestimulatory composition. Alternatively, the response may comprise aT-helper or CTL-based response to one or more of the epitopes providedin the immune stimulatory composition. All three of these responses mayoriginate from naïve or memory cells. In other embodiments, a“protective effective amount” of an immune stimulatory composition is anamount which, when administered to a subject, is sufficient to conferprotective immunity upon the subject.

Immunize: To render a subject protected from an infectious disease, suchas by vaccination.

Isolated: An “isolated” or “purified” biological component (such as anucleic acid, peptide, protein, protein complex, or virus-like particle)has been substantially separated, produced apart from, or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, that is, other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” or “purified” thus include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids or proteins. The term “isolated” or “purified” does notrequire absolute purity; rather, it is intended as a relative term.Thus, for example, an isolated biological component is one in which thebiological component is more enriched than the biological component isin its natural environment within a cell, or other production vessel.Preferably, a preparation is purified such that the biological componentrepresents at least 50%, such as at least 70%, at least 90%, at least95%, or greater, of the total biological component content of thepreparation.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compositions, such as one or more flavivirus vaccines, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a protein, polypeptide, or peptide.

A conservative substitution in a polypeptide is substitution of oneamino acid residue in a protein sequence for a different amino acidresidue having similar biochemical properties. Typically, conservativesubstitutions have little to no impact on the activity of a resultingpolypeptide. For example, a flavivirus protein including one or moreconservative substitutions (for example no more than 2, 5, 10, 20, 30,40, or 50 substitutions) retains the structure and function of thewild-type protein. A polypeptide can be produced to contain one or moreconservative substitutions by manipulating the nucleotide sequence thatencodes that polypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. In one example, such variants can bereadily selected by testing antibody cross-reactivity or its ability toinduce an immune response. Examples of conservative substitutions areshown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Premembrane (prM) protein: A flavivirus structural protein. The prMprotein is an approximately 25 kDa protein that is the intracellularprecursor for the membrane (M) protein. prM is believed to stabilize theE protein during transport of the immature virion to the cell surface.When the virus exits the infected cell, the prM protein is cleaved tothe mature M protein, which is part of the viral envelope (Reviewed inLindenbach and Rice, In: Fields Virology, Knipe and Howley, eds.,Lippincott, Williams, and Wilkins, 991-1041, 2001).

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity of oneor more signs or symptoms of a disease.

Recombinant: A recombinant nucleic acid, protein or virus is one thathas a sequence that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or by the artificial manipulation of isolated segments ofnucleic acids, for example, by genetic engineering techniques. The termrecombinant includes nucleic acids, proteins and viruses that have beenaltered solely by addition, substitution, or deletion of a portion of anatural nucleic acid molecule, protein or virus.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. If the two compared sequences sharehomology, then the designated output file will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals (such as mice, rats, rabbits,sheep, horses, cows, and non-human primates).

Therapeutically effective amount: A quantity of a specified agent (suchas an immunogenic composition) sufficient to achieve a desired effect ina subject being treated with that agent. For example, this may be theamount of a virus vaccine useful for eliciting an immune response in asubject and/or for preventing infection by the virus. In the context ofthe present disclosure, a therapeutically effective amount of a denguevirus vaccine, for example, is an amount sufficient to increaseresistance to, prevent, ameliorate, and/or treat infection caused by adengue virus in a subject without causing a substantial cytotoxic effectin the subject. The effective amount of a dengue virus immunestimulating composition useful for increasing resistance to, preventing,ameliorating, and/or treating infection in a subject will be dependenton, for example, the subject being treated, the manner of administrationof the therapeutic composition and other factors.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of infectious or other types of disease. The immunogenicmaterial may include attenuated or killed microorganisms (such asbacteria or viruses), or antigenic proteins (including VLPs), peptidesor DNA derived from them. An attenuated vaccine is a virulent organismthat has been modified to produce a less virulent form, but neverthelessretains the ability to elicit antibodies and cell-mediated immunityagainst the virulent form. A killed vaccine is a previously virulentmicroorganism that has been killed with chemicals or heat, but elicitsantibodies against the virulent microorganism. Vaccines may elicit bothprophylactic (preventative) and therapeutic responses. Methods ofadministration vary according to the vaccine, but may includeinoculation, ingestion, inhalation or other forms of administration.Vaccines may be administered with an adjuvant to boost the immuneresponse.

Vector: A vector is a nucleic acid molecule allowing insertion offoreign nucleic acid without disrupting the ability of the vector toreplicate and/or integrate in a host cell. A vector can include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. An insertional vector is capable of insertingitself into a host nucleic acid. A vector can also include one or moreselectable marker genes and other genetic elements. An expression vectoris a vector that contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes.

Virus-like particle (VLP): Virus particles made up of one of more viralstructural proteins, but lacking the viral genome. Because VLPs lack aviral genome, they are non-infectious. In some embodiments, the VLPs areflavivirus VLPs, such as dengue VLPs. In particular examples, flavivirusVLPs include two flavivirus structural proteins—prM and E.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Introduction

Dengue viruses (DENV) are the most important mosquito transmitted viralpathogens infecting humans. Nearly half of the world's population livesin DENV endemic regions and millions of infections and many thousands ofdeaths occur annually. DENV infection produces a spectrum of disease,most commonly causing a self-limiting flu-like illness known as denguefever, yet with increased frequency manifesting as life-threateningdengue hemorrhagic fever (DHF). Dengue immune responses are bothprotective and pathogenic as immunity to one of the four dengueserotypes can enhance subsequent infection with another serotype toproduce DHF. Decades of effort to develop safe and efficacious denguevaccines remain unsuccessful, primarily due to imbalanced immunity andconcern that such imbalances could leave vaccinees not only unprotectedbut with increased susceptibility to enhanced disease.

Described herein is the development of a DENV serotype 1 (DENV-1) DNAvaccine with the immunodominant cross-reactive epitopes associated withimmune enhancement removed. Comparison of the wild-type with thiscross-reactivity reduced (CRR) vaccine demonstrated that both vaccinesare equally protective against lethal homologous virus challenge. Underconditions simulating natural exposure during the inter-vaccine boostperiod prior to acquiring balanced protective immunity, wild-typevaccinated mice enhanced a normally sub-lethal heterologous DENV-2infection resulting in DHF-like disease and 95% mortality. However, CRRvaccinated mice did not produce immunodominant cross-reactive enhancingantibodies, they exhibited redirected serotype-specific and protectiveimmunity, and significantly reduced morbidity and mortality notdiffering from naïve mice in response to sub-lethal challenge. Thus,these data demonstrate that in an in vivo DENV disease model,non-protective vaccine-induced immunity can prime vaccinees for enhancedDHF-like disease and that CRR DNA immunization significantly reducesthis vaccine safety concern. The sculpting of immune memory by themodified vaccine and resulting redirection of immunity hold greatpromise for developing novel vaccine prime-boost strategies againstmulti-strain pathogens such as DENV where vaccines remain elusive.

IV. Overview of Several Embodiments

Disclosed herein are dengue virus E-glycoprotein polypeptides containingmutations that eliminate immunodominant cross-reactive epitopesassociated with immune enhancement. In some cases, the dengue virusE-glycoproteins further include mutations that introduce a strong CD4 Tcell epitope. The disclosed E-glycoprotein polypeptides, or nucleic acidmolecules encoding the polypeptides, can be used, for example, inmonovalent or tetravalent vaccines against dengue virus. In someembodiments, the dengue virus E-glycoprotein is not a DENV-2E-glycoprotein.

Provided herein are cross-reactivity reduced dengue virus E-glycoproteinpolypeptides having amino acid substitutions at residues correspondingto positions 106, 107, 310 and 311, and either position 364 or position389 of dengue serotype 1 (DENV-1) E-glycoprotein (SEQ ID NO: 1). Theprovided E-glycoprotein polypeptides optionally further includemutations corresponding to positions 468, 478, 482 and 487 of DENV-1E-glycoprotein (SEQ ID NO: 1). The mutations at positions 468, 478, 482and 487 of SEQ ID NO: 1 occur in the C-terminal region of the proteinwhich is derived from JEV.

In some embodiments, provided herein is an isolated cross-reactivityreduced dengue virus E-glycoprotein polypeptide, wherein the polypeptideis selected from (1) a dengue serotype 1 virus (DENV-1) E-glycoproteinpolypeptide comprising an arginine at position 106, an aspartic acid atposition 107, an aspartic acid at position 310, a lysine at position311, a glutamine at position 364, an isoleucine at position 468, athreonine at position 478, a valine at position 482 and a leucine atposition 487, numbered with reference to SEQ ID NO: 1; (2) a dengueserotype 2 virus (DENV-2) E-glycoprotein polypeptide comprising anarginine at position 106, an aspartic acid at position 107, an glutamicacid at position 310, an arginine at position 311, an arginine atposition 364, an isoleucine at position 468, a threonine at position478, a valine at position 482 and a leucine at position 487, numberedwith reference to SEQ ID NO: 2; (3) a dengue serotype 3 virus (DENV-3)E-glycoprotein polypeptide comprising an aspartic acid at position 106,an aspartic acid at position 107, an aspartic acid at position 308, alysine at position 309, an aspartic acid at position 362, an isoleucineat position 466, a threonine at position 476, a valine at position 480and a leucine at position 485, numbered with reference to SEQ ID NO: 3;and (4) a dengue serotype 4 virus (DENV-4) E-glycoprotein polypeptidecomprising a glutamic acid at position 106, an aspartic acid at position107, an aspartic acid at position 310, a glutamine at position 311, aglutamine at position 389, an isoleucine at position 468, a threonine atposition 478, a valine at position 482 and a leucine at position 487,numbered with reference to SEQ ID NO: 4.

The mutations at positions 468, 478, 482 and 487 of SEQ ID NO: 1;positions 466, 476, 480 and 485 of SEQ ID NO: 3; and positions 468, 478,482 and 487 of SEQ ID NO: 4 occur in the C-terminal region of eachprotein, which is derived from JEV (see Example 1).

In particular embodiments, the amino acid sequence of the polypeptide isat least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 1, wherein the polypeptide comprises an arginineat position 106, an aspartic acid at position 107, an aspartic acid atposition 310, a lysine at position 311, a glutamine at position 364, anisoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487 of SEQ ID NO: 1. In someexamples, the amino acid sequence of the polypeptide comprises SEQ IDNO: 1.

In other particular embodiments, the amino acid sequence of thepolypeptide is at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 2, wherein the polypeptidecomprises an arginine at position 106, an aspartic acid at position 107,an glutamic acid at position 310, an arginine at position 311, anarginine at position 364, an isoleucine at position 468, a threonine atposition 478, a valine at position 482 and a leucine at position 487 ofSEQ ID NO: 2. In some examples, the amino acid sequence of thepolypeptide comprises SEQ ID NO: 2.

In other particular embodiments, the amino acid sequence of thepolypeptide is at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 3, wherein the polypeptidecomprises an aspartic acid at position 106, an aspartic acid at position107, an aspartic acid at position 308, a lysine at position 309, anaspartic acid at position 362, an isoleucine at position 466, athreonine at position 476, a valine at position 480 and a leucine atposition 485 of SEQ ID NO: 3. In some examples, the amino acid sequenceof the polypeptide comprises SEQ ID NO: 3.

In yet other particular embodiments, the amino acid sequence of thepolypeptide is at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 4, wherein the polypeptidecomprises a glutamic acid at position 106, an aspartic acid at position107, an aspartic acid at position 310, a glutamine at position 311, aglutamine at position 389, an isoleucine at position 468, a threonine atposition 478, a valine at position 482 and a leucine at position 487 ofSEQ ID NO: 4. In some examples, the amino acid sequence of comprises SEQID NO: 4.

Also provided are isolated dengue virus-like particles (VLPs) comprisingthe cross-reactivity reduced dengue virus E-glycoprotein disclosedherein. In some embodiments, the VLP further includes a dengue virus prMprotein. The VLP can optionally further include a dengue virus Cprotein.

Further provided are recombinant nucleic acid molecules encoding across-reactivity reduced dengue virus E-glycoprotein disclosed herein,or encoding a VLP containing a cross-reactivity reduced dengue virusE-glycoprotein. In some embodiments, the recombinant nucleic acidmolecule comprises a nucleotide sequence at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12. In particular examples, the recombinant nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11 or SEQ ID NO: 12.

Also provided are vectors comprising the recombinant nucleic acidmolecules disclosed herein, and isolated cells comprising such vectors.

Compositions, such as immune stimulating compositions, are furtherprovided by the present disclosure. In some embodiments, thecompositions include a cross-reactivity reduced dengue virusE-glycoprotein polypeptide, a VLP comprising a cross-reactivity reduceddengue virus E-glycoprotein polypeptide, a recombinant nucleic acidmolecule encoding a cross-reactivity reduced dengue virus E-glycoproteinpolypeptide, or a vector encoding a cross-reactivity reduced denguevirus E-glycoprotein polypeptide or encoding a VLP comprising thecross-reactivity reduced polypeptide, and a pharmaceutically acceptablecarrier. In some embodiments, the composition further includes anadjuvant.

Further provided is a tetravalent dengue virus vaccine. In someembodiments, the tetravalent dengue virus vaccine comprises a pluralityof recombinant nucleic acid molecules, each encoding a cross-reactivityreduced dengue virus E-glycoprotein polypeptide from a different denguevirus serotype. In some examples, the tetravalent dengue virus vaccinecomprises a recombinant nucleic acid molecule encoding the DENV-1polypeptide of SEQ ID NO: 1, a recombinant nucleic acid moleculeencoding the DENV-2 polypeptide of SEQ ID NO: 2, a recombinant nucleicacid molecule encoding the DENV-3 polypeptide of SEQ ID NO: 3, and arecombinant nucleic acid molecule encoding the DENV-4 polypeptide of SEQID NO: 4.

The present disclosure also provides methods of eliciting an immuneresponse in a subject against dengue virus. In some embodiments, themethod includes administering to the subject a therapeutically effectiveamount of a polypeptide, VLP, nucleic acid molecule, vector, compositionor vaccine as disclosed herein. In some embodiments, the subject is amammal, such as a human.

Also provided herein are compositions comprising a DENV-1 E-glycoproteinpolypeptide, a DENV-2 E-glycoprotein polypeptide, a DENV-3E-glycoprotein polypeptide and a DENV-4 E-glycoprotein polypeptide; or arecombinant nucleic acid molecule encoding the DENV-1 E-glycoproteinpolypeptide, a recombinant nucleic acid molecule encoding the DENV-2E-glycoprotein polypeptide, a recombinant nucleic acid molecule encodingthe DENV-3 E-glycoprotein polypeptide and a recombinant nucleic acidmolecule encoding the DENV-4 E-glycoprotein polypeptide, wherein:

(1) the DENV-1 E-glycoprotein polypeptide comprises an arginine atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a lysine at position 311, a glutamine at position 364, anisoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO: 1;

(2) the DENV-2 E-glycoprotein polypeptide comprises an arginine atposition 106, an aspartic acid at position 107, an glutamic acid atposition 310, an arginine at position 311, an arginine at position 364,an isoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO: 2;

(3) the DENV-3 E-glycoprotein polypeptide comprises an aspartic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 308, a lysine at position 309, an aspartic acid at position362, an isoleucine at position 466, a threonine at position 476, avaline at position 480 and a leucine at position 485, numbered withreference to SEQ ID NO: 3; and/or

(4) the DENV-4 E-glycoprotein polypeptide comprises a glutamic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a glutamine at position 311, a glutamine at position 389,an isoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO: 4.

In some embodiments, provided herein is a composition in which (1) theamino acid sequence of the DENV-1 polypeptide is at least 95%, at least96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:1, and wherein the polypeptide comprises an arginine at position 106, anaspartic acid at position 107, an aspartic acid at position 310, alysine at position 311, a glutamine at position 364, an isoleucine atposition 468, a threonine at position 478, a valine at position 482 anda leucine at position 487 of SEQ ID NO: 1; (2) the amino acid sequenceof the DENV-2 polypeptide is at least 95%, at least 96%, at least 97%,at least 98% or at least 99% identical to SEQ ID NO: 2, and wherein thepolypeptide comprises an arginine at position 106, an aspartic acid atposition 107, an glutamic acid at position 310, an arginine at position311, an arginine at position 364, an isoleucine at position 468, athreonine at position 478, a valine at position 482 and a leucine atposition 487 of SEQ ID NO: 2; (3) the amino acid sequence of the DENV-3polypeptide is at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to SEQ ID NO: 3, and wherein the polypeptidecomprises an aspartic acid at position 106, an aspartic acid at position107, an aspartic acid at position 308, a lysine at position 309, anaspartic acid at position 362, an isoleucine at position 466, athreonine at position 476, a valine at position 480 and a leucine atposition 485 of SEQ ID NO: 3; and/or (4) the amino acid sequence of theDENV-4 polypeptide is at least 95%, at least 96%, at least 97%, at least98% or at least 99% identical to SEQ ID NO: 4, and wherein thepolypeptide comprises a glutamic acid at position 106, an aspartic acidat position 107, an aspartic acid at position 310, a glutamine atposition 311, a glutamine at position 389, an isoleucine at position468, a threonine at position 478, a valine at position 482 and a leucineat position 487 of SEQ ID NO: 4.

In particular examples, the DENV-1 polypeptide comprises the amino acidsequence of SEQ ID NO: 1, the DENV-2 polypeptide comprises the aminoacid sequence of SEQ ID NO: 2, the DENV-3 polypeptide comprises theamino acid sequence of SEQ ID NO: 3 and/or the DENV-4 polypeptidecomprises the amino acid sequence of SEQ ID NO: 4.

Further provided is a method of eliciting an immune response in asubject against dengue virus by administering to the subject atherapeutically effective amount of a composition comprising a DENV-1E-glycoprotein polypeptide, a DENV-2 E-glycoprotein polypeptide, aDENV-3 E-glycoprotein polypeptide and a DENV-4 E-glycoproteinpolypeptide; or a recombinant nucleic acid molecule encoding the DENV-1E-glycoprotein polypeptide, a recombinant nucleic acid molecule encodingthe DENV-2 E-glycoprotein polypeptide, a recombinant nucleic acidmolecule encoding the DENV-3 E-glycoprotein polypeptide and arecombinant nucleic acid molecule encoding the DENV-4 E-glycoproteinpolypeptide. In some examples, the method includes administering a DNAvaccine (such as a vaccine comprising recombinant nucleic acid moleculesencoding cross-reactivity reduced dengue virus E-glycoproteins from eachof DENV-1, DENV-2, DENV-3 and DENV-4), followed by administering aprotein booster vaccine (such as a vaccine including recombinant,inactivated virus, or live attenuated virus).

V. Cross-Reactivity Reduced Dengue Virus E-Glycoprotein Polypeptides

Provided by the present disclosure are cross-reactivity reduced denguevirus E-glycoprotein polypeptides in which immunodominant epitopesassociated with immune enhancement have been removed by select aminoacid substitutions. In particular, for each serotype of dengue virus,amino acid substitutions were made at residues corresponding topositions 106, 107, 310, 311 and 364 of DENV-1 (SEQ ID NO: 1), with theexception that the DENV-4 E-glycoprotein includes a mutation at position389 instead of position 364. The cross-reactivity reducedE-glycoproteins provided herein were based on extensive mutagenesisstudies to select the best mutation site as measured by the highestreduction on complex cross-reactive monoclonal antibody binding. Thesestudies revealed that for DENV-4, mutation at residue 389 reducedantibody binding to a much greater degree than mutation at position 364(which is located structurally very close to residue 389). Thus,mutation at 389 was selected for this serotype. In addition, because theE-glycoprotein of DENV-3 is two amino acids shorter than the E proteinfrom the remaining serotypes, the mutations in the DENV-3 E-glycoproteinoccur at positions 106, 107, 308, 309 and 362 (SEQ ID NO: 3).

In some instances, the amino acid substituted for each dengue serotypeat each position varies. The specific residues at each position wereselected to enhance or maintain antigen secretion.

In some embodiments, the dengue virus E-glycoprotein polypeptidesfurther contain a potent CD4 T cell epitope identified in thetransmembrane domain of the E-glycoprotein of West Nile virus. To insertthe CD4 T cell epitope, mutations were made at residues 468 (valine toisoleucine), 478 (alanine to threonine), 482 (threonine to valine) and487 (valine to leucine) for DENV-1, DENV-2 and DENV-4 E protein (SEQ IDNOs: 1, 2 and 4), which corresponded to 466 (valine to isoleucine), 476(alanine to threonine), 480 (threonine to valine) and 485 (valine toleucine) for DENV-3 E protein.

Provided herein are dengue virus E-glycoprotein polypeptides comprisingthe following mutations:

DENV-1 DENV-2 DENV-3 DENV-4 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQID NO: 4 G106R G106R G106D G106E L107D L107D L107D L107D K310D K310EK308D K310D E311K E311R E309K E311Q P364Q P364R P362D L389Q V468I V468IV466I V468I A478T A478T A476T A478T T482V T482V T480V T482V V487L V487LV485L V487L

In some embodiments of the present disclosure, the dengue virusE-glycoprotein is at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identicalto SEQ ID NO: 1, wherein the polypeptide comprises an arginine atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a lysine at position 311, a glutamine at position 364, anisoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487 of SEQ ID NO: 1; or is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 2,wherein the polypeptide comprises an arginine at position 106, anaspartic acid at position 107, an glutamic acid at position 310, anarginine at position 311, an arginine at position 364, an isoleucine atposition 468, a threonine at position 478, a valine at position 482 anda leucine at position 487 of SEQ ID NO: 2; or is at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% identical to SEQ ID NO: 3, wherein the polypeptidecomprises an aspartic acid at position 106, an aspartic acid at position107, an aspartic acid at position 308, a lysine at position 309, anaspartic acid at position 362, an isoleucine at position 466, athreonine at position 476, a valine at position 480 and a leucine atposition 485 of SEQ ID NO: 3; or is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to SEQ ID NO: 4, and wherein the polypeptide comprises aglutamic acid at position 106, an aspartic acid at position 107, anaspartic acid at position 310, a glutamine at position 311, a glutamineat position 389, an isoleucine at position 468, a threonine at position478, a valine at position 482 and a leucine at position 487 of SEQ IDNO: 4.

In specific non-limiting examples, the amino acid sequence of thecross-reactivity reduced dengue virus E-glycoprotein comprises SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

VI. Immunostimulatory Compositions and Administration Thereof

The immunostimulatory compositions provided herein can include, forexample, a cross-reactivity reduced dengue virus E-glycoproteinpolypeptide, a recombinant nucleic acid molecule encoding across-reactivity reduced dengue virus E-glycoprotein polypeptide, a VLPcomprising a cross-reactivity reduced dengue virus E-glycoproteinpolypeptide, or a recombinant nucleic acid molecule (such as a vector)encoding a VLP. In some cases, the immunostimulatory compositions aremonovalent vaccines for dengue virus (i.e. contain E-glycoprotein from asingle serotype of dengue virus). In other instances, theimmunostimulatory compositions are tetravalent vaccines for dengue virus(i.e. contain E-glycoprotein from all four dengue virus serotypes).

The cross-reactivity reduced dengue virus E-glycoprotein polypeptidesand VLPs (including nucleic acid molecules encoding the cross-reactivityreduced dengue virus polypeptides and VLPs) disclosed herein can be usedas dengue virus vaccines to elicit an immune response, such as aprotective immune response, against dengue virus.

The provided immunostimulatory dengue virus polypeptides, constructs orvectors encoding such polypeptides, are combined with a pharmaceuticallyacceptable carrier or vehicle for administration as an immunestimulatory composition to human or animal subjects. In a particularembodiment, the immune stimulatory composition administered to a subjectdirects the synthesis of a dengue virus E-glycoprotein as describedherein, and a cell within the body of the subject, after incorporatingthe nucleic acid within it, secretes VLPs comprising the E-glycoprotein.It is believed that such VLPs then serve as an in vivo immunestimulatory composition, stimulating the immune system of the subject togenerate protective immunological responses. In some embodiments, morethan one immune stimulatory dengue virus polypeptide, construct orvector may be combined to form a single preparation.

The immunogenic formulations may be conveniently presented in unitdosage form and prepared using conventional pharmaceutical techniques.Such techniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing adose or unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations encompassed herein mayinclude other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use as immunestimulatory compositions, may be administered through different routes,such as oral, including buccal and sublingual, rectal, parenteral,aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical.They may be administered in different forms, including but not limitedto solutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route ofadministration. By way of example, intramuscular injections may rangefrom about 0.1 ml to about 1.0 ml. Those of ordinary skill in the artwill know appropriate volumes for different routes of administration.

Immune stimulatory compounds (for example, vaccines) can be administeredby directly injecting nucleic acid molecules encoding peptide antigens(broadly described in Janeway & Travers, Immunobiology: The ImmuneSystem In Health and Disease, page 13.25, Garland Publishing, Inc., NewYork, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42-45, 1996).Vectors that include nucleic acid molecules described herein, or thatinclude a nucleic acid sequence encoding a dengue virus E-glycoproteinpolypeptide may be utilized in such DNA vaccination methods.

Thus, the term “immune stimulatory composition” as used herein alsoincludes nucleic acid vaccines in which a nucleic acid molecule encodinga cross-reactivity reduced dengue virus E-glycoprotein polypeptide isadministered to a subject in a pharmaceutical composition. For geneticimmunization, suitable delivery methods known to those skilled in theart include direct injection of plasmid DNA into muscles (Wolff et al.,Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specificprotein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989),co-precipitation of DNA with calcium phosphate (Benvenisty and Reshef,Proc. Natl. Acad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes(Kaneda et al., Science 243:375, 1989), particle bombardment (Tang etal., Nature 356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791,1993), and in vivo infection using cloned retroviral vectors (Seeger etal., Proc. Natl. Acad. Sci. 81:5849, 1984). Similarly, nucleic acidvaccine preparations can be administered via viral carrier.

The amount of immunostimulatory compound in each dose of an immunestimulatory composition is selected as an amount that induces animmunostimulatory or immunoprotective response without significant,adverse side effects. Such amount will vary depending upon whichspecific immunogen is employed and how it is presented. Initialinjections may range from about 1 μg to about 1 mg, with someembodiments having a range of about 10 μg to about 800 μg, and stillother embodiments a range of from about 25 μg to about 500 μg. Followingan initial administration of the immune stimulatory composition,subjects may receive one or several booster administrations, adequatelyspaced. Booster administrations may range from about 1 μg to about 1 mg,with other embodiments having a range of about 10 μg to about 750 μg,and still others a range of about 50 μg to about 500 μg. Periodicboosters at intervals of 1-5 years, for instance three years, may bedesirable to maintain the desired levels of protective immunity.

In some embodiments, a subject receives a DNA prime vaccination, forexample, a nucleic acid molecule (such as a vector) encoding a denguevirus E-glycoprotein polypeptide described herein, and subsequentlyreceives a protein boost vaccination (such as recombinant, inactivatedvirus, or live attenuated virus). DNA prime-protein boost strategies fordengue virus are discussed further in section IX below.

Dengue virus polypeptides or VLPs (or nucleic acid molecules encodingdengue virus polypeptides or VLPs), or compositions thereof, areadministered in any suitable manner, such as with pharmaceuticallyacceptable carriers. Pharmaceutically acceptable carriers are determinedin part by the particular composition being administered, as well as bythe particular method used to administer the composition. Accordingly,there is a wide variety of suitable formulations of pharmaceuticalcompositions of the present disclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Particular methods for administering nucleic acid molecules are wellknown in the art. In some examples, the nucleic acid encoding the denguevirus polypeptide or VLP is administered by injection (such asintramuscular or intradermal injection) or by gene gun.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time, or to inhibit or prevent flavivirus infection. Thedose required will vary from subject to subject depending on thespecies, age, weight and general condition of the subject, the severityof the infection being treated, the particular composition being usedand its mode of administration. An appropriate dose can be determined byone of ordinary skill in the art using only routine experimentation.

The pharmaceutical or immune stimulatory compositions or methods oftreatment may be administered in combination with other therapeutictreatments. For example, the compositions provided herein can beadministered with an adjuvant, such as Freund incomplete adjuvant orFreund's complete adjuvant.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host.

VII. Immune Enhancement Following Heterologous DENV Exposure

The most significant risk factor for severe dengue disease is secondaryinfection with a heterologous serotype (Murphy and Whitehead, Annu RevImmunol 29:587-619, 2011). Two distinct yet not mutually exclusivemechanisms for immune enhancement upon heterologous exposure explain thepathogenic manifestations characterizing DHF/DSS. The leading hypothesisis that DHF occurs via antibody-dependent enhancement (ADE) ofinfection, a phenomenon originally described for flaviviruses but laterfound to occur with diverse pathogens (Hawkes, Aust J Exp Biol Med Sci42:465-482, 1964; Halstead, Rev Infect Dis 11 Suppl 4:S830-839, 1989;Morens, Clin Infect Dis 19:500-512, 1994; Thomas et al., Expert RevVaccines 5:409-412, 2006). Preexisting cross-reactive antibodies from aprevious infection (or maternal antibodies in infants) recognize andbind to heterologous virus in a secondary infection, yet are unable toneutralize this virus, either because they are non-neutralizing, or fora lack of sufficient avidity or occupancy (Pierson et al., Cell HostMicrobe 4:229-238, 2008). However, these non-neutralizing antibody-viruscomplexes increase the infection of monocytes via their Fc receptors,dramatically increasing viral replication and load, thereby causing DHF.

Moreover, recent studies additionally implicate an important role foraltered intracellular signaling enhancing ADE effects by suppressinginnate antiviral protein responses (Thomas et al., Expert Rev Vaccines5:409-412, 2006; Mahalingam and Lidbury, Proc Natl Acad Sci USA99:13819-13824, 2002). Weakly- and non-neutralizing cross-reactiveantibodies induced from immunodominant B-cell epitopes are known to makeup the majority of the humoral immune response to DENV infection (Lai etal., J Virol. 82(13):6631-6643, 2008; Crill et al., PLoS ONE 4:e4991,2009). Although there are now numerous reports of in vitro ADE of DENVinfection, ADE has not been demonstrated to occur in humans or to causeDHF, yet recent studies provide strong support for an in vivo role forADE in human DHF. The majority of the rare cases of primary DHFinfections occur in infants born to DENV immune mothers and in one studythe age at which infants acquired DHF correlated with predicted age ofmaximal in vitro enhancement (Kliks et al., Am J Trop Med Hyg38:411-419, 1988). A prospective study conducted in Vietnam indicatedthat the in vitro enhancing capability of maternally derived infantantibody in pre-exposure sera correlated with subsequent DENV diseaseseverity (Chau et al., J Infect Dis 198:516-524, 2008). In a recentlydeveloped type I/type II IFN receptor knock-out mouse model (AG129) ofDENV disease (Williams et al., Ann NY Acad Sci 1171 Suppl 1:E12-23,2009), sub-neutralizing levels of homologous or heterologous DENV immunemouse sera (Zellweger et al., Cell Host Microbe 7(2):128-139, 2010;Balsitis et al., PLoS Pathog 6:e1000790, 2010) or cross-reactive humanMAbs (Beltramello et al., Cell Host Microbe 8:271-283, 2010) enhancedviral replication in vivo producing a DHF-type disease.

The second, related mechanism of immune enhanced DENV pathology, also ageneralized phenomenon, posits that there is a highly skewed cellularresponse to heterologous DENV infection driven by low affinity,cross-reactive memory CD4+ and CD8+ T cells (Brehm et al., Nat Immunol3:627-634, 2002; Welsh and Selin, Nat Rev Immunol 2:417-426, 2002; Welshet al., Annu Rev Immunol 22:711-743, 2004). During primary DENVinfection cross-reactive and serotype-specific T cell clones areselected for increasing affinity to the infecting DENV serotype. Uponheterologous infection these pre-existing memory T cells with reducedaffinity for the secondarily infecting serotype dominate the immuneresponse relative to higher affinity T cells specific for the currentlyinfecting virus (Rothman, J Clin Invest 113:946-951, 2004). Thedomination of lower affinity clones in secondary response appears to bedue to both the preferential expansion of cross-reactive T cellsoriginated from the primary infection and from apoptosis ofhigh-affinity secondarily stimulated T cells induced by the highantigenic loads associated with secondary infection (Combadiere et al.,J Exp Med 187:349-355, 1998; Mongkolsapaya et al., Nat Med 9:921-927,2003). Both processes result in T cell populations inefficient at viralclearance.

Moreover, additional evidence suggests that such low affinityanamnestically stimulated T cells contribute to DENV immunopathology bytheir continued secretion of pro-inflammatory cytokines and vasoactivemediators causing the increased vascular permeability and hemorrhagicmanifestations characterizing DHF/DSS (Welsh and Selin, Nat Rev Immunol2:417-426, 2002; Mangada and Rothman, J Immunol 175:2676-2683, 2005;Mongkolsapaya et al., J Immunol 176:3821-3829, 2006). Cross-reactive Tcell epitopes have been identified across the DENV proteome, however,immunodominant CD8+ T cell epitopes in non-structural protein NS3 appearto be those most strongly associated with DHF (Mathew and Rothman,Immunol Rev 225:300-313, 2008; Friberg et al., Immunol Cell Biol89(1):122-129, 2010; Duangchinda et al., Proc Natl Acad Sci USA107(39):16922-16927, 2010).

The common theme throughout DENV immune enhancement is the concept of‘original antigenic sin’ originally describing a phenomenon observed inresponse to influenza, it has since been found to be a generalizedphenomenon (Francis, Ann Intern Med 39:203-221, 1953; Welsh andFujinami, Nat Rev Microbiol 5:555-563, 2007). Original antigenic sindescribes the shift in the immunodominance hierarchy that occurs whenprior exposure to cross-reactive antigens alters and inhibits subsequentimmune response to related antigens, either in new infections or throughvaccination (Brehm et al., Nat Immunol 3:627-634, 2002). Both humoraland cellular responses can be plagued by such misdirected orinappropriate heterotypic immunity (Welsh and Fujinami, Nat RevMicrobiol 5:555-563, 2007). The phenomenon is most severe when thecross-reactive antigenic responses are immunodominant as is the casewith both cellular and humoral DENV immunity. Thus, for DENV not onlydoes original antigenic sin appear to play an important role in the moresevere DENV pathologies but it also creates a vaccine safety issue as alack of protective tetravalent vaccine induced immunity could primevaccinees for DHF upon subsequent infection (Murphy and Whitehead, AnnuRev Immunol 29:587-619, 2011). Such caution is valid as a similarsituation occurred when children immunized with inactivated respiratorysyncytial virus vaccine suffered increased mortality when naturallyexposed to RSV and the increased morbidity and mortality appeared to bedue to both ADE and T cell induced cytokine immunopathology (Thomas etal., Expert Rev Vaccines 5:409-412, 2006; Kim et al., Am J Epidemiol89:422-434, 1969).

Concerns for dengue vaccination priming individuals for severe diseasevia immune enhancement have necessitated the postulates that denguevaccines must produce balanced, tetravalent, and protective immunity,optimally derived from a single dose providing long-lasting protection(Miller, Curr Opin Mol Ther 12:31-38, 2010; Murphy and Whitehead, AnnuRev Immunol 29:587-619, 2011). Because of the past experience of robust,long-term immunity derived from live yellow fever 17D vaccine andothers, the majority of public health efforts have focused on thedevelopment of live-attenuated dengue vaccines (Durbin and Whitehead,Curr Top Microbiol Immunol 338:129-143, 2009). Despite advancements overthe past three decades, dengue vaccine development continues to facesignificant road blocks challenging the necessity of these historicalpostulates. Inducing a balanced, tetravalent, and protective immunityhas been the biggest hurdle and remains largely unresolved in the timespans considered necessary, typically ≦3 months, the duration of the lowtransmission season in many endemic countries (Sun et al., Hum Vaccin5:33-40, 2009; Morrison et al., J Infect Dis 201:370-377, 2010).Tetravalent live-replicating vaccines are susceptible to bothreplication and immune interference between different vaccine serotypes(Guy et al., Am J Trop Med Hyg 80:302-311, 2009). This phenomenon isboth a biological and a stochastic process since the target cell numberin the injection site is significantly lower than the total infectiousvirions in tetravalent DENV vaccine formulations. Resulting imbalancesin serotype-specific immunity exacerbate concerns of vaccine-inducedimmune enhancement, regardless of its mechanistic basis, and thisconcern has not been addressed by existing dengue vaccine candidates inclinical trials (Miller, Curr Opin Mol Ther 12:31-38, 2010; Whitehead etal., Nat Rev Microbiol 5:518-528, 2007; Hatch et al., IDrugs 11:42-45,2008).

The inventors have developed a vaccine platform specifically addressingthese obstacles to DENV vaccine development. The DNA vaccine platformconsists of an expression plasmid containing only the envelope (E, theprimary protective antigen) and premembrane (prM) structural proteingenes. Upon uptake by host cells the structural genes are transcribedand translated and the proteins self-assemble into virus-like particles(VLPs) which are antigenically indistinguishable from virus (Chang etal., Ann NY Acad Sci 951:272-285, 2011; Chang et al., Virology306:170-180, 2003). The wild-type (WT) flavivirus DNA vaccines aredemonstrably safe, immunogenic, and protective against Japaneseencephalitis, West Nile, and dengue viruses in both non-human animals(Chang et al., Virology 306:170-180, 2003; Mohageg et al., Opt Express15:4869-4875, 2007; Davis et al., J Virol 75:4040-4047, 2001; Chang etal., Vaccine 25:2325-2330, 2007) and in humans (Martin et al., J InfectDis 196:1732-1740, 2007). DNA vaccines have a number of advantages overother vaccine platforms. They can stimulate strong CD4+ T cell responsesas do inactivated and subunit vaccines and yet also strongly stimulateCD8+ T cell responses similar to live attenuated vaccines (Martin etal., J Infect Dis 196:1732-1740, 2007; Laylor et al., Clin Exp Immunol117:106-112, 2009). Because VLPs lack infectious RNA and arenon-replicating, tetravalent dengue DNA vaccines do not suffer from thereplication interference obstacles impeding the live-attenuated vaccineapproaches (Laylor et al., Clin Exp Immunol 117:106-112, 2009; Petersenand Roehrig, J Infect Dis 196:1721-1723, 2007). Most importantly, unlikelive viruses or the inactivated vaccines made from them, DNA vaccinescan be readily manipulated and engineered to prime specific epitopes andredirect immune response away from immunodominant, pathogenic B cell andT cell epitopes. This sculpted immune memory priming can redirectsubsequent vaccine boosts or natural exposure toward protective,DENV-specific epitopes increasing both vaccine safety and efficacy.

The present disclosure tackles the previously unaddressed concern ofvaccine-induced predisposition to severe dengue disease via immuneenhancement. Described is the introduction of substitutions into twodistinct E protein antigenic regions of a DENV-1 DNA vaccine (pVD1)ablating cross-reactive, immunodominant, weakly or non-neutralizing Bcell epitopes associated with immune enhancement (Crill et al., PLoS ONE4:e4991, 2009), thereby reducing the potential for this cross-reactivityreduced vaccine (pVD1-CRR) to potentiate DHF via ADE. These CRR denguevaccines are also less susceptible to heterologous immune enhancementvia cross-reactive T cell induced “cytokine storm” mechanisms. Becausethey contain only the prM and E proteins, they lack entirely thewell-characterized, immunodominant, DHF-associated CD 8+ T cell epitopeslocated in the NS3 protein. The results disclosed herein demonstratethat by priming the immune system with CRR dengue DNA vaccines, theimmune landscape can be sculpted to increase protective DENV-specificneutralizing antibody responses and avoid anamnestic responses tocross-reactive enhancing epitopes that are inherent to other DENVvaccine approaches. In addition to DENV-1, corresponding mutations weremade in the E-glycoproteins of DENV-2, DENV-3 and DENV-4; the sequencesof these proteins are also provided herein.

VIII. Redirecting the Immune Response to DENV Vaccination

The present disclosure describes construction of wild-type (WT) andcross-reactivity reduced (CRR) DENV-1 DNA vaccine candidates anddemonstrates that both WT and CRR vaccines stimulate similar levels ofneutralizing antibodies. Moreover, immunized mice were equally protectedfrom homologous DENV-1 virus challenge. The CRR vaccine candidate wasspecifically engineered to reduce the potential for vaccine-inducedsusceptibility to severe dengue disease, a theoretical safety concernthat has hampered dengue vaccine development for decades (Murphy andWhitehead, Annu Rev Immunol 29:587-619, 2011). These results confirm, inan in vivo dengue disease model, that this vaccine safety issue is realand indicate that the disclosed CRR vaccine approach can resolve thislong-standing, previously intractable roadblock to dengue vaccinedevelopment. It is demonstrated that by disrupting immunodominant,pathogenic epitopes, the immunodominance hierarchy can be redirectedaway from pathogenic toward normally subdominant, protective epitopes.Under conditions simulating natural exposure during theinter-vaccination boost period prior to achieving balanced protectiveimmunity, WT vaccinated mice enhanced a heterologous, self-limiting,normally sub-lethal DENV-2 infection into a DHF-like disease pathologyresulting in 95% mortality. CRR vaccinated mice however, lackedvaccine-induced immunodominant enhancing antibodies, exhibited increaseddominance of protective, neutralizing immunoglobulins, and hadsignificantly reduced morbidity and mortality that did not differ fromnaïve mice.

The finding of vaccine-induced severe dengue disease and mortality viaADE is consistent with and supported by two recent studies ofADE-induced dengue disease in AG129 mice using passive transfer of DENVimmune sera (Zellweger et al., Cell Host Microbe 7(2):128-139, 2010;Balsitis et al., PLoS Pathog 6:e1000790, 2010). Using the same DENV-2S221 virus as in the present study, these studies found thatadministering 10⁴, 10⁵, or 10⁶ pfu of DENV-2 S221 was sub-lethal tonaïve AG129 mice receiving passively transferred normal mouse serum 24hours prior to challenge. However, mice receiving heterologous DENV-1,-3, or -4 immune sera 24 hours prior to challenge died 4-5 DPC fromsevere dengue disease resembling DHF. The disease pathology observed inthese studies was similar to that seen in human DHF and includedvascular leak related pathology of the visceral organs,thrombocytopenia, elevated levels of pro-inflammatory cytokines,increased viremia, hepatitis, and increased virus replication in liversinusoidal endothelial cells. Confirmation that this enhanced DHF-likedisease and mortality resulted from ADE was supported by passivetransfer of well-characterized enhancing EDII_(FP) MAbs, and by rescuefrom enhanced mortality by passive transfer of an engineered EDII_(FP)MAb unable to bind FcγR, thereby demonstrating that the enhanced diseasepathology required FcγR interaction (Balsitis et al., PLoS Pathog6:e1000790, 2010). In the present study, it was discovered that whenexposed to normally sub-lethal doses of DENV-2 S221, pVD1-WT vaccinatedmice suffered 95% mortality from ADE-induced DHF-like disease and thatthe enhanced disease pathology and mortality could be significantlyreduced by immunization with a modified pVD1-CRR vaccine that did notstimulate immunodominant cross-reactive antibodies.

In humans, severe dengue disease is associated with 10-100 foldincreases in viral loads compared to DF patients (Murphy and Whitehead,Annu Rev Immunol 29:587-619, 2011). A 100-fold greater increase inDENV-2 viremia of pVD1-WT vaccinated mice compared to pVD1-CRRvaccinated mice was observed. This 100-fold increase was similar to thatobserved in experimental models of ADE in Rhesus monkeys (Goncalvez etal., Proc Natl Acad Sci USA 104:9422-9427, 2007), 5-35 times higher thanin passively transferred AG129 mice (Zellweger et al., Cell Host Microbe7(2):128-139, 2010; Balsitis et al., PLoS Pathog 6:e1000790, 2010), and10-fold greater than the increase in DHF patients relative to DFpatients in a Thai study (Vaughn et al., J Infect Dis 181:2-9, 2000).

The present study also revealed a rapid induction of diverse DENV-2neutralizing antibody populations by pVD1-CRR vaccinated mice inresponse to heterologous DENV-2 infection. It was hypothesized that therapid rise in DENV-2 neutralizing antibody titers was due to anincreased memory response to complex cross-reactive neutralizingantibodies recognizing epitopes not altered in the CRR vaccine, such asMAb 1B7, and to increased primary induction of DENV-2 serotype-specificpotently neutralizing antibodies such as 9A3D-8 and 3H5. One explanationis that WT vaccinated mice would not have the immunological energyavailable to invest in such sub-dominate neutralizing antibody responsesbecause of their immunodominant production of EDII_(FP) and EDIII_(CR)antibodies (original antigenic sin) whereas CRR vaccinated mice shouldhave increased induction of such neutralizing antibody populationsspecifically because they lacked EDII_(FP) and EDIII_(CR) antibodypriming. There was a very large increase in 1B7-like antibodies forCRR-vaccinated mice relative to WT vaccinated mice, supporting theimportance of this class of neutralizing antibody. However, the majorityof the large increase in DENV-2 neutralization by CRR vaccinated miceappears to be due to DENV-2 serotype-specific potently neutralizingantibody.

The concern of inappropriate vaccine-induced immunodominant antibodyresponses in dengue vaccinology and the approach described herein ofgenetically modifying these pathogenic epitopes to redirect theimmunodominance hierarchy has parallels with other variable strainpathogens such as HIV and influenza. Both HIV and influenza vaccinologysuffer from the plague of original antigenic sin and strain-specificimmunodominance. For all of these viruses, producing efficaciousvaccines requires altering the native, wild-type immune responses. Inboth HIV and influenza there has been much recent interest in this areaof modifying immunogens to redirect immune responses, typically referredto as immune dampening and immune refocusing (Tobin et al., Vaccine26:6189-6199, 2008). Some generalizations from this body of work areconsistent with and support the CRR DENV DNA vaccination results. Thedampening of immunodominant epitopes resulted in decreased induction ofantibodies recognizing the targeted epitopes while increasing the amountof antibody stimulated from natively sub-dominant epitopes. Moreover, inspite of such major alterations in immunodominance hierarchies,antigenically modified immunogens induced similar total overall antibodytiters as did WT immunogens. Together, these results imply that via avariety of approaches it is possible to successfully redirectvaccine-induced immune responses to sculpt original antigenic sin andimprove safety and/or efficacy without altering overall vaccineimmunogenicity.

IX. DNA Prime-Protein Boost Vaccine Strategies

There is substantial interest in utilizing heterologous vaccineprime-boost strategies to improve and broaden immunogenicity, especiallyin the context of DNA vaccination (Dale et al., Methods Mol Med127:171-197, 2006; Guenaga et al., PLoS ONE 6:e16074, 2011; Ding et al.,PLoS ONE 6:e16563, 2011; Chen et al., J Virol 81:11634-11639, 2007;Simmons et al., Virology 396:280-288, 2010). In this context,‘heterologous’ typically refers to the use of different vaccine formatsto present the same viral immunogen between prime and boost, mostcommonly DNA prime and protein (recombinant, inactivated virus, or liveattenuated virus) boost. Much of this interest has also been directedtoward HIV (Walker and Burton, Curr Opin Immunol 22:358-366, 2010) andinfluenza (Ding et al., PLoS ONE 6:e16563, 2011; Wei et al., Science329:1060-1064, 2010) where the vaccine goal is to increase the breadthof neutralization and hence protection from these highly variablemulti-strain pathogens. Because of the similarities to DENV and thedifficulties of rapidly inducing balanced tetravalent immunity withcurrent DENV live attenuated vaccines (Murphy and Whitehead, Annu RevImmunol 29:587-619, 2011), DNA prime-protein boost strategies areparticularly appealing to DENV vaccinology. Chen et al. comparedmonovalent DENV-1 prM/E DNA vaccination with that of a Venezuelan equineencephalitis viral replicon particle (VRP) delivering DENV-1 prM/Eproteins in non-human primates. They found that DNA prime followed byVRP boost produced higher DENV-1 total IgG and neutralizing antibodytiters than either DNA-DNA or VRP-VRP prime-boost strategies. Moreover,only the DNA-VRP prime-boost prevented viremia following DENV-1challenge (Chen et al., J Virol 81:11634-11639, 2007). Simmons et al.examined tetravalent DNA, inactivated virus, and live attenuated DENVprime-boost schemes in non-human primates (Simmons et al., Virology396:280-288, 2010). They found that tetravalent inactivated virus primeand tetravalent live attenuated virus (TLAV) boost produced the highestELISA and neutralizing antibody titers and prevented detectable viremiafollowing challenge while still inducing strong anamnestic increases inneutralizing antibody.

It was found that DENV-1 CRR DNA vaccination redirected subsequentimmunity in response to DENV-2 challenge and increased the induction ofa broad repertoire of neutralizing antibodies to produce a rapid, largemagnitude DENV-2 neutralizing response with increasedcross-neutralization to other DENV serotypes. These findings suggestthat CRR DNA vaccines hold great potential for novel DENV vaccinestrategies that take advantage of the benefits of both DNA and liveattenuated virus vaccines. One such strategy is to prime hosts with alow-dose tetravalent CRR DNA vaccine to induce a balanced memoryresponse with limited neutralizing antibody to each serotype. Thispriming should allow for efficient TLAV boost 1-2 months later torapidly produce long-lasting, balanced, and protective tetravalentimmunity with reduced virus enhancing capabilities. This vaccinestrategy takes advantage of the strong T cell priming properties of DNAvaccines, the potent induction of long-lasting high-titer antibodyresponses of LAVs, and the improved safety and protective efficacyprofile of the CRR modifications disclosed herein.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Materials and Methods

This example describes the materials and experimental procedures usedfor the studies described in Example 2.

Vaccine Construction, Characterization, and Preparation

The construction of pVD1-WT and pVD1-CRR vaccine plasmids was the sameas for DENV-2 DNA vaccine plasmids and has been previously described indetail (Crill et al., PLoS ONE 4:e4991, 2009; Chang et al., Virology306:170-180, 2003). Briefly, pVD1-WT was constructed using DENV-1 strain(56)BC94/95 for cDNA cloning of premembrane (prM) and 80% DENV-1envelope (E) with the C terminal 20% of the E protein replaced with thehomologous region of Japanese encephalitis virus (JEV). 80% DENV-1 Ecorresponds to the ectodomain and the C terminal 20% corresponds to thehelical stems (E-H1, E-H2) and transmembrane domain (TMD) anchor helices(E-T1, E-T2) (Zhang et al., Nat Struct Biol 10:907-912, 2003). Inaddition the pVD1-WT vaccine utilized in this study, and as the templatefor constructing pVD1-CRR, contained a potent CD4+ T cell TMD epitopeidentified in West Nile virus (WNV). The inclusion of this epitope intothe DENV-2 DNA vaccine (pVD2) and into pVD2-CRR boosts the neutralizingantibody responses of vaccinated mice about 2-fold. Introduction of theWNV TMD CD4+ epitope into pVD1 required introducing four amino acidsubstitutions (V474I, A484T, T488V, and V493L) into the C terminal 20%JEV sequence of pVD1.

pVD1-CRR was constructed using pVD1-WT as the DNA template and theprimers listed in Table 1, and with Stratagene Quick Change®multi-site-directed mutagenesis kit (Stratagene, La Jolla, Calif.)following the manufacturer's recommended protocols. Structural generegions and regulatory elements of all plasmids were sequenced entirelyupon identification of the correct mutation. Automated DNA sequencingwas performed using a Beckman Coulter CEQ™ 8000 genetic analysis system(Beckman Coulter, Fullerton, Calif.) and analyzed using Beckman CoulterCEQ™ 8000 (Beckman Coulter) and Lasergene® software (DNASTAR, Madison,Wis.). Vaccine DNA for immunization was prepared using Qiagen endo-freemaxi-prep kits.

Mice—Protective Efficacy

Six-week old type I/type II IFN receptor-knock-out mice (AG129) wereused. AG129 mice (DVBD, n=26) were immunized with 100 μg of pVD1-WT orpVD1-CRR vaccine (50 μg in each thigh i.m.) at 0, 4, and 8 weeks.Immunized and naïve age-matched mice (n=8) were challenged at 12 weeksintraperitoneally (i.p.) with 1.1×10⁵ FFU of DENV-1 Mochizuki strain.Based upon results from previous dose-titration experiments, fouranimals from each vaccine treatment and two naïve mice were a prioriselected for sacrifice 3 and 8 days post challenge (DPC). Sick animalsreaching predetermined morbidity endpoints were anesthetized, totalblood was collected via cardiac puncture, euthanized and carcasses werefrozen at −80° C. for subsequent analysis of brains.

Mice—Vaccine Safety

AG129 mice (DVBD, n=26) were immunized with 100 μg of either pVD1-WT orpVD1-CRR vaccine (50 μg in each thigh, i.m.) at 0 weeks. Immunized andage-matched naïve mice (n=10) were challenged at 12 weeks (i.p.) with4.2×10⁵ FFU of DENV-2 S221 (Zellweger et al., Cell Host Microbe7(2):128-139, 2010). Four mice from each vaccine treatment and two naïvemice were a priori selected for sacrifice 3 DPC. Sick animals reachingpre-determined morbidity endpoints were anesthetized, total blood wascollected via cardiac puncture, euthanized, and necropsy was performedwith livers collected and stored in 10% formalin for histology.

Histology

Liver tissue sections were prepared, stained, and analyzed. IHC utilizedVentana Medical Systems ultraView Universal Alkaline Phosphatase RedDetection Kit following the manufacturer's recommended protocols and aBenchmark Ultra automated system. Pretreatment was with protease II for6 minutes, rabbit anti DENV-2 NS 1 serum was used at a 1:20 dilution,and anti-rabbit secondary antibody was applied for 12 minutes.

Characterization of pVD1-Cross-Reactivity Reduced (CRR) Plasmid VaccineInduced Virus-Like Particle (VLP) Antigen

Antigen-capture ELISA (Ag-ELISA) was used to characterize VLP antigenfollowing previously described protocols (Crill et al., PLoS ONE4:e4991, 2009). Briefly, VLP antigen was collected from mutagenized andWT pVD1 transformed COS-1 cells. Secreted antigen was captured in theinner 60 wells of Immulon II HB flat-bottom 96-well plates (DynatechIndustries, Inc., Chantilly, Va.) with polyclonal rabbit anti-DENV-1 WTVLP sera, incubated overnight at 4° C., and wells were blocked with 300μl of StartBlock blocking buffer (Pierce, Rockford, Ill.) according tothe manufacturer's recommended protocol. Antigen was diluted 2-fold inPBS, incubated for 2 hours at 37° C. and detected with murinehyper-immune ascitic fluid (MHIAF) specific for DENV-2 diluted in 5%milk/PBS. MHIAF was detected using horseradish peroxidase conjugatedgoat anti-mouse IgG (Jackson ImmunoResearch, Westgrove, Pa.) in 5%milk/PBS and incubated for 1 hour at 37° C. Bound conjugate was detectedwith 3,3′5,5′-tetramethylbenzidine substrate (TMB; Neogen Corp.,Lexington, Ky.), the reaction was stopped with 2N H₂SO₄ and measured atA₄₅₀ using a Synergy HT Multi-Detection Microplate Reader (Bio-TekInstruments, Inc., Winooski, Va.). WT and mutant antigens were screenedagainst the MAb panel using the same ELISA protocol as above with theexception that 2-fold dilutions of the specific MAb replaced theanti-DENV-2 MHIAF and antigens were used at a single standardizedconcentration producing an optical density (OD) of 1.0. Standardizedconcentrations of WT and mutant VLP antigens were analyzed in Ag-ELISAto determine MAb end point reactivities.

Monoclonal Antibodies

MAbs 4G2, 6B6C-1, 4A1B-9, 1B7, D3-5C9-1, 1A1D-9, 9D12, 9A3D-8, 3H5, andD2-1F1-3 were obtained from hybridomas in the collection of theArbovirus Diseases Branch, DVBD, CDC. Many of these MAbs originated fromthe work of John Roehrig (Roehrig et al., Virology 246:317-328, 1998),4G2, 5H3, 1B7, D3-5C9-1, 9D12, 3H5, and D2-1F1-3 hybridomas wereoriginally obtained by the CDC from the Walter Reed Army Institute(Henchal et al., Am J Trop Med Hyg 34:162-169, 1985). MAbs 23-1, 23-2,and 5-2 were provided by Dr. L.- K. Chen of Tzu Chi University, Hualien,Taiwan. MAbs 10-D35A, 20-783-745014, and MDVP-55A are commerciallyavailable and were purchased from Fitzgerald Industries International,GenWay Biotech Inc., and Immunology Consultants Laboratory Inc.,respectively.

Epitope-Specific IgG ELISA (ES-ELISA)

We used DENV-1 and DENV-2 ES-ELISA to determine total DENV-1 or DENV-2IgG end-point titers and to determine DENV-1 or DENV-2 EDII_(FP),EDIII_(CR), EDII_(FP)EDIII_(CR), and non-EDII_(FP)EDIII_(CR)epitope-specific IgG percentages. The DENV-2 ES-ELISA has beenpreviously described (Crill et al., PLoS ONE 4:e4991, 2009) and theDENV-1 ES-ELISA was similarly used with a few modifications. Briefly,rabbit anti pVD1 or pVD2 sera was coated onto plates overnight at 4° C.Standardized concentrations of WT and CRR antigens were captured, AG129mouse serum was diluted four-fold down the plate, and detected with goatanti-mouse IgG (Jackson ImmunoResearch). Vaccinated mouse sera werediluted 1:100-1:400 and serially titrated. The DENV-1 epitope-specificknock-out antigens utilized are described in Table 2. OD values weremodeled as non-linear functions of the log₁₀ sera dilutions using anon-linear sigmoidal dose-response (variable slope) regression in GraphPad Prism version 4.0 and endpoint titrations were determined as thetiter where the OD value equaled two-times the OD value of the testserum reacted against normal COS-1 antigen.

EDII_(FP), EDIII_(CR), EDII_(FP)EDIII_(CR), and non-EDII_(FP)EDIII_(CR)epitope-specific IgG percentages were calculated as previously described(Crill et al., PLoS ONE 4:e4991, 2009) with minor modifications. TheDENV-1 epitope-specific IgG percentages for all vaccinated mice werecalculated by dividing the IgG endpoint titer obtained with eachknock-out antigen (EDII_(FP), EDIII_(CR), or EDII_(FP)EDIII_(CR)) by theendpoint titer obtained with pVD1-WT antigen on the same sera,subtracting this value from 1.0, and multiplying by 100. Specifically,EDII_(FP) epitope-specific percentages were calculated as100×[1.0−(DENV-1 EDII_(FP) antigen endpoint/DENV-1 WT antigenendpoint)]. EDIII_(CR) epitope-specific percentages were calculated as100×[1.0−(DENV-1 EDIII_(CR) antigen endpoint/DENV-1 WT antigenendpoint)]. EDII_(FP)EDIII_(CR) IgG was calculated as 100×[1.0−(DENV-1EDII_(FP)EDIII_(CR) antigen endpoint/DENV-1 WT antigen endpoint)].

Non-EDII_(FP)EDIII_(CR) IgG proportions were calculated as 100*(endpointEDII_(FP)EDIII_(CR)/endpoint WT) for WT vaccinated mice and becausepVD1-CRR vaccinated mouse sera contain antibodies that do not recognizesome WT antigen epitopes (WT antigen acts as the knock-out antigen) butrecognize the modified epitopes of the CRR antigens, the pVD1-CRRantigen (EDII_(FP)EDIII_(CR)) replaced pVD1-WT antigen to determine 100%E reactivity for pVD1-CRR vaccinated mice and similarly, thenon-EDII_(FP)EDIII_(CR) IgG proportion for pVD1-CRR immunized mice wascalculated as 100*(DENV-1 WT antigen endpoint/DENV-1 EDII_(FP)EDIII_(CR)antigen endpoint). DENV-2 epitope specific IgG populations weresimilarly determined but used the previously described DENV-2 EDII_(FP),EDIII_(CR), and EDII_(FP)EDIII_(CR) knock out antigens to determineendpoints (Crill et al., PLoS ONE 4:e4991, 2009) for both pVD1-WT andpVD1-CRR vaccinated sera. In cases where the endpoint titer determinedwith a mutant antigen was the same or greater than the endpoint titerobtained with the cognate antigen, it was interpreted as undetectablelevels of antibody recognizing the epitope of interest and the percentof IgG was arbitrarily set at 0.05%.

Epitope-Blocking ELISA

Epitope blocking ELISA was utilized to determine the vaccinated mouseresponse to well-characterized murine MAb epitopes. This ELISA was setup similar to ES-ELISA in that plates were coated overnight at 4° C.with rabbit anti-DENV-2 serum, blocked with StartBlock (Pierce), driedand WT DENV-2 VLP antigen was captured 1 hour at 37° C. After washing,pVD1-WT or pVD1-CRR vaccinated, DENV-2 challenged mouse serum (3 DPC,pooled for each vaccine treatment) was diluted 1:40 in wash buffer andincubated 1 hour at 37° C. Following pooled serum incubation and wash,0.5 μg of horseradish peroxidase (HRP)-conjugated MAb was added to eachwell and incubated for 1 hour at 37° C. to compete with the alreadybound antibody from vaccinated mouse serum for WT DENV-2 VLP antigen.Bound conjugate was detected with TMB substrate and plates incubated for10 minutes prior to being stopped with H₂SO₄ and ODs were determined asOD=OD of A₄₅₀−OD of A₆₃₀. Percent blocking was determined by comparisonto replicate wells with 0.5 μg HRP labeled MAb competing againstpre-adsorbed normal mouse serum. Percent blocking was determined usingthe following calculation, 100−100*[(OD of vaccinated serum on DENV-2Ag−OD of vaccinated serum on normal Ag)/OD of normal serum on DENV-2Ag−OD of normal serum on normal Ag)].

Antibody-Dependent Enhancement

Heat inactivated pVD1-WT and pVD1-CRR vaccinated mouse sera (12 weekspost vaccination) were pooled into 4 groups for each vaccine treatment,diluted, and titrated. Virus (DENV-2 16681) was added to each dilutionand incubated for 1 hour at 37° C. K562 cells (MOI=0.5) were added tothe antibody-virus complexes and incubated 2 hours. After infection,cells were centrifuged, supernatants removed, resuspended in RPMI media(10% FBS) and plated. DENV infection alone was used as virus control.

Focus Reduction Micro-Neutralization Assay (FRμNT)

FRμNT assay was utilized as previously described (Crill et al., PLoS ONE4:e4991, 2009) with few modifications. Vaccinated mouse sera werediluted 1:10, heat inactivated, titrated 2-fold to the volume of 40 μL,and 320 virus focus-forming units (FFU)/40 μL (DENV-1 56BC94/95, DENV-216681, DENV-3 116RC1396, or DENV-4 130) was added to each dilution.FRμNT titers were calculated for each virus relative to a backtitration. Exact 50% of FRμNT titers were modeled using Graph Pad Prismversion 4 sigmoidal dose response (variable slope) non-linearregression. Values are the average of two independent replicates.

Viremia and Brain Virus Titers

Viremia was determined in a similar antigen-focus assay as described forFRμNT except that no virus was added. Cells were incubated, overlaid,acetone fixed, immunostained and counted as described for FRμNT. Virusbrain titers were determined from previously frozen mouse carcasses.Brain tissue was aspirated with a 3 mL syringe and 18 gauge needle,weighed, and resuspended in 200 μL BA-1 media. 50 μL was used in theFRμNT as described. FFU/g of tissue was back calculated from theaspirated brain mass.

Cytokine Assays

Serum samples from terminally ill mice were frozen at −70° C., thawed onice, and virus was heat inactivated. Serum samples were used indetection of cytokines in a mouse inflammatory cytometric bead array (BDBiosciences, San Jose, Calif.) as per manufactures protocol.Fluorescence was detected on BD FACSCalibur and analyzed with FCAP arraysoftware (BD Biosciences, San Jose, Calif.).

Example 2 Redirecting the Immune Response to DENV Vaccination

This example describes the development and characterization of across-reactivity reduced (CRR) DENV-1 DNA vaccine.

Vaccine Construction

Based upon previously published and unpublished results with dengueviruses (DENV) and other flaviviruses (Crill et al., PLoS ONE 4:e4991,2009), specific substitutions were introduced into the envelope proteinof a DENV-1 premembrane/envelope protein (prM/E) expression vectorplasmid (Chang et al., Virology 306:170-180, 2003) and a DENV-1 DNAvaccine candidate with reduced ability to induce the cross-reactiveantibodies associated with antibody-dependent enhancement (ADE) wasgenerated. The DENV-1 wild-type DNA vaccine (pVD1-WT) utilized as thetemplate for the cross-reactivity reduced vaccine (pVD1-CRR) in thisstudy was based upon extensive studies with DENV-2 vaccines (Chang etal., Virology 306:170-180, 2003). Substitutions were introduced into twoimportant antigenic regions of the DENV E protein, the highly conservedfusion peptide of structural domain II (EDII_(FP)) and thereceptor-binding immunoglobulin-like domain III (EDIII; FIG. 1).

EDII_(FP) contains multiple overlapping immunodominant B cell epitopesinducing broadly cross-reactive, weakly or non-neutralizing antibodiesassociated with antibody enhanced severe DENV disease in both mice andhumans (Lai et al., J Virol. 82(13):6631-6643, 2008; Crill et al., PLoSONE 4:e4991, 2009; Zellweger et al., Cell Host Microbe 7(2):128-139,2010; Balsitis et al., PLoS Pathog 6:e1000790, 2010; Beltramello et al.,Cell Host Microbe 8:271-283, 2010; Crill and Chang, J Virol78:13975-13986, 2004; Stiasny et al., J Virol 80:9557-9568, 2006). EDIIIcontains two well-characterized overlapping antigenic regions, onestimulating DENV complex cross-reactive antibodies varying in theirneutralizing capabilities (Roehrig et al., Virology 246:317-328, 1998;Sukupolvi-Petty et al., J Virol 81:12816-12826, 2007; Gromowski et al.,J Virol 82:8828-8837, 2008) and the other stimulating DENVserotype-specific, potently neutralizing antibodies associated with DENVserotype-specific immunity (Crill et al., PLoS ONE 4:e4991, 2009;Beltramello et al., Cell Host Microbe 8:271-283, 2010; Sukupolvi-Pettyet al., J Virol 81:12816-12826, 2007; Gromowski and Barrett, Virology366:349-360, 2007).

Multiple substitutions at EDII_(FP) residues G106 and L107, and at K310,E311, and P364 in the cross-reactive antigenic region of EDIII(EDIII_(CR); FIG. 1), were examined. Final individual substitutions atthese five residues were selected based upon their influence on in vitrovirus-like particle (VLP) secretion and their effect on the reactivitiesof a panel of well-characterized monoclonal antibodies (MAbs, Table 2).EDII_(FP) substitutions tended to increase VLP secretion and knocked outthe reactivity of flavivirus group and sub-group cross-reactive MAbs.EDIII_(CR) substitutions were specifically selected not to interferewith the binding of potently neutralizing EDIII serotype-specific MAbs.These substitutions tended to reduce VLP secretion relative to WT andablated the reactivity predominately of DENV sub-complex cross-reactiveMAbs. The final pVD1-CRR plasmid, containing substitutions at all fiveof the sites across both EDII_(FP) and EDIII_(CR) antigenic regions(FIG. 1), knocked-out or reduced the reactivity to below detectablelevels of 11 cross-reactive monoclonal antibodies in the panel (Table2). The cross-reactive MAbs whose reactivities were not significantlyreduced were 1B7, a sub-group cross-reactive MAb that neutralizes all 4DENV serotypes (Table 3), and 10-D35A and D3-5C9-1, weakly (for DENV-2)and non-neutralizing DENV complex cross-reactive MAbs respectively.

pVD1-WT and pVD1-CRR Vaccines Both Induce High Titer TotalImmunoglobulin and Neutralizing Antibody, Protecting Mice from LethalDENV-1 Challenge

To test for vaccine protective efficacy, WT and CRR DENV-1 DNA vaccineswere compared by immunizing AG129 mice and subsequently challenging witha lethal dose of homologous DENV-1 (Mochizuki strain). AG129 Type I/TypeII IFN receptor knock-out mice have impaired neutralizing antibodyresponses (Schijns et al., J Immunol 153:2029-2037, 1994) and asexpected our DNA vaccines were not as immunogenic in these mice as inimmunocompetent mouse strains. Therefore, the standard immunizationschedule was altered from a single 100 μg boost 4 weeks followingprimary vaccination (100 μg) to two 100 μg boosts at 4 and 8 weeks. Thisschedule produced immune responses approaching the magnitude of thosepreviously observed in other mouse strains with the two dose schedule(Chang et al., Virology 306:170-180, 2003). Mice were challenged at 12weeks. Prior to challenge, mouse sera was collected and vaccine-inducedimmune responses were measured.

ES-ELISA (Crill et al., PLoS ONE 4:e4991, 2009) was used to determinetotal DENV-1 IgG and DENV-1 EDII_(FP) IgG. Total DENV-1 IgG end-pointtiters were similar between the two vaccines and averaged7.7×10⁴±1.5×10⁴ and 7.5×10⁴±1.8×10⁴ for pVD1-WT and pVD1-CRR immunizedmice respectively (p=0.524; FIG. 2A). However, pVD1-CRR immunized micehad significantly lower proportions of EDII_(FP) IgG than did pVD1-WTimmunized mice, averaging 2.1±0.96% and 33±6.4% of the total IgGresponse respectively (p=0.0005, FIG. 2B). Only two pVD1-CRR immunizedmice had measurable EDII_(FP) IgG (4.8% and 22%) with the remaining micebeing below detectable levels (conservatively set to 1.0% forstatistical analyses and EDII_(FP) IgG titer calculations). Theproportion of EDII_(FP) IgG for WT immunized mice was large andvariable, ranging from 0.8% to 73%, similar to that observed in DENVinfected humans (Stiasny et al., 2006; Crill et al., 2009; Beltramelloet al., 2010). Calculated EDII_(FP) IgG end-point titers averaged2.0×10⁴±4.6×10³ and 1.1×10³±3.4×10² for WT and CRR immunized micerespectively (p=0.0001, FIG. 2C). 50% neutralization (Nt₅₀) titers,measured by FRμNT, averaged 91.1 and 48.8 for pVD1-WT and pVD1-CRRvaccinated mice respectively (p=0.0047; FIG. 2D). The lower Nt₅₀ titerfor pVD1-CRR immunized mice was likely due to reduced induction ofEDIII_(CR) antibodies recognizing epitopes similar to those ofneutralizing MAbs 1A1D-2 and 9D12 that lost all measurable reactivityfor the pVD1-CRR tissue culture derived VLPs (Table 1). These resultsindicate that targeted substitution within EDII_(FP) reduces theimmunodominance of this region.

Age-matched naïve (18 week old, n=8), pVD1-WT and pVD1-CRR (n=26 each)vaccinated animals were challenged with 1.1×10⁵ focus forming units(FFU) of the mouse-brain adapted DENV-1 Mochizuki strain. This dose wasgreater than 100 LD₅₀ for 6-8 week old mice. All immunized mice except asingle animal from each vaccine treatment were protected and survivedchallenge (94% survival) which was highly significant in comparison tonaïve mice (25% survival, p=0.0003; FIG. 2E). Time to death of naïvemice ranged from 8-17 days post challenge (DPC) and averaged 11.7 DPC;the single vaccinated animals died 7 and 8 DPC for WT and CRR vaccinetreatments respectively. Surviving mice showed no signs of sickness.Although DENV-1 Mochizuki is a mouse-brain adapted virus, limitedneurological symptoms such as paralysis in terminally sick mice wasobserved, and most exhibited hunched and ruffled posture, lethargy, anda lack of interest in food and water leading to weight loss prior tobeing euthanized. Four mice from each vaccine treatment and two naïvemice were scheduled a priori to be euthanized at 3 DPC, a time pointprior to any outward sign of disease, and 8 DPC, a few days after theinitial signs of morbidity and mortality. Vaccinated mice exhibited100-fold and 10-fold lower viremia 3 and 8 DPC respectively, compared tonaïve controls; yet there was no difference in mean viremic titersbetween WT or CRR vaccinated mice at either time point (FIG. 2F). Atwo-way ANOVA found vaccine treatment to be highly significant(P<0.0001) and to account for 68% of the variation. Bonferroni posttestsindicated that viremia of each vaccinated group was significantly lowerthan for naïve mice 3 DPC (p<0.001) whereas only pVD1-CRR immunizedmouse viremia was significantly lower than naïve mice 8 DPC (p<0.01).

Because DENV-1 Mochizuki strain is a mouse-brain adapted virus, virustiters of mouse brain homogenates were also determined. Brains from bothnaïve and vaccinated mice were all negative 3 DPC (<50 FFU/g braintissue). By 8 DPC, virus titers of naïve mouse brain tissue was 100-1000times greater than for vaccinated mice, however limited sampling andhigh variance precluded rigorous statistical conclusions. The single 8DPC naïve mouse brain virus titer was 2.3×10⁴ FFU/g, median virus titersof WT and CRR vaccinated mice (n=4 each) were 50 and 325 FFU/g of braintissue respectively with three of four WT and two of four CRR vaccinatedbrain tissue titers below the limits of assay detection (<50 FFU/gtissue). A one sample t-test of all 8 vaccinated mouse brain titerscompared to the single 2.3×10⁴ titer for the naïve mouse stronglyrejected the null hypothesis (p=0.0042), suggesting that there was lessvirulence in vaccinated compared to naïve mouse brains.

Consistent with an anamnestic response to DENV-1 challenge, both WT andCRR immunized surviving mice exhibited similar order of magnitudeincreases in Nt₅₀ titer 28 DPC (GMT=690, 95% CI 504-944 and =362, 95% CI218-601 for WT and CRR respectively). Together, these data suggest thatpVD1-WT and pVD1-CRR vaccines are similarly immunogenic and able toinduce protective immunity against lethal homologous DENV-1 challenge inthe AG129 DENV vaccine model.

Cross-Reactivity Reduced Vaccine Redirects Immunity from ImmunodominantPathological Responses Toward Protective Responses to Improve VaccineSafety and Efficacy

To test for the possibility of an improved safety profile of the CRRDENV-1 DNA vaccine, AG129 mice were immunized with 100 μg WT or CRRvaccine, and after waiting 84 days for potential transientcross-protection to dissipate, mice were challenged with a sub-lethaldose of DENV-2. This scenario mimics the vaccine safety concern ofenhanced disease that could occur following DENV exposure withincomplete tetravalent vaccine seroconversion, e.g., prior to finalboosting with some prolonged vaccine schedules (Sun et al., Hum Vaccin5:33-40, 2009; Morrison et al., J Infect Dis 201:370-377, 2010).Immunized mice were challenged with DENV-2 S221, a mouse adapted isolateof Taiwanese strain PL046 (Shresta et al., J Virol 80:10208-10217,2006). This virus is capable of producing lethal hemorrhagic disease inAG129 mice, similar to that observed in human DHF, via ADE whensub-protective levels of DENV immune sera are passively transferred tomice and subsequently challenged with non-lethal doses of DENV-2 S221(Zellweger et al., Cell Host Microbe 7(2):128-139, 2010; Balsitis etal., PLoS Pathog 6:e1000790, 2010). Based on previous work with CRRDENV-2 DNA vaccines, it was hypothesized that pVD1-WT mice would besusceptible to antibody-enhanced DENV disease due to high levels ofvaccine-induced cross-reactive antibodies and that upon subsequentheterologous challenge, antibodies recognizing these immunodominantepitopes would be stimulated anamnestically. Conversely, pVD1-CRRvaccinated mice would lack such immunodominant cross-reactive antibodypriming and secondary responses, be less susceptible to antibodyenhanced viral replication, and have increased immunological capabilityto rapidly respond with appropriate DENV-2 immunity. pVD1-CRR vaccinatedmice should therefore be less susceptible to antibody enhanced DENV-2disease.

DENV-1 CRR Vaccinated Mice have Reduced Levels of ImmunodominantEDII_(FP) IgG and Reduced Dengue Disease Mortality Compared to DENV-1 WTVaccinated Mice Following DENV-2 Challenge

To assess if pVD1-WT and -CRR vaccines induce different antibodyrepertoires in vaccinated mice, 12 week post-vaccination sera wascollected and utilized in ES-ELISA (Crill et al., PLoS ONE 4:e4991,2009) to determine vaccine induced DENV-1 IgG titers and the proportionsand titers of IgG recognizing EDII_(FP) epitopes (FIG. 1). There was nodifference in total DENV-1 IgG GMT between pVD1-WT (3168, 95% CI1792-5600; n=20) and pVD1-CRR immunized mice (1429, 95% CI 621-3289,n=23; p=0.128, two-tailed Mann-Whitney U test; FIG. 3A). The percent oftotal DENV-1 IgG recognizing EDII_(FP) epitopes averaged 17.7±4.6% and2.56±1.2% for pVD1-WT and pVD1-CRR immunized mice respectively(p=0.0009). Again, only two CRR immunized mice had detectable levels ofEDII_(FP) IgG (9.4% and 21%) with all remaining mice having nomeasurable EDII_(FP) IgG (=1.0% for statistical analyses; FIG. 3B). Thecalculated DENV-1 IgG GMT recognizing EDII_(FP) epitopes wassignificantly lower for CRR (38.6, 95% CI 19.4-76.9) than for WTimmunized mice (272, 95% CI 97.2-760, p=0.0023, one-tailed Mann-WhitneyU test; FIG. 3C). These results again indicate that pVD1-WT and pVD1-CRRvaccines are similarly immunogenic overall, but that the pVD1-CRRvaccine induces less IgG recognizing immunodominant EDII_(FP) epitopes.

Age-matched naïve (n=10), pVD1-WT, and pVD1-CRR (n=26 each) immunizedmice were challenged (i.p.) with a sub-lethal dose (4.2×10⁵ FFU) ofDENV-2 S221 12 weeks following immunization for vaccine treatmentgroups. All naïve mice survived DENV-2 challenge, though they didexhibit signs of morbidity including decreased activity, coat ruffling,and weight loss. pVD1-WT vaccinated mice suffered from severely enhancedDENV-2 pathology, including hemorrhagic manifestations producing 95%mortality (p<0.0001 compared to naïve). Conversely, pVD1-CRR vaccinatedmice exhibited reduced DENV-2 disease enhancement and 68% survival whichdid not differ from naïve mouse survival (100%, p=0.0769) and yet wasmuch greater than WT vaccinated mouse survival (4.5%, p<0.0001, theBonferroni multiple comparison adjusted a=0.017; FIG. 3D). Sick micebegan exhibiting external signs of morbidity late on day 3 and reachedterminal endpoints 4.5-5.5 DPC for both groups with one WT vaccinatedmouse surviving to 8 DPC.

To characterize which humoral immune determinates might be responsiblefor the reduced disease severity and mortality of CRR vaccinated mice,sera from 3, 4, and 5 DPC was examined. Four animals in each vaccinetreatment were selected a priori for sacrifice 3 DPC, a time point whenmice exhibited no outward signs of morbidity. Four and 5 DPC sera wereobtained from terminally sick mice via cardiac puncture just prior toeuthanasia. Thus, sample sizes were limited on some of these days forpVD1-CRR vaccinated mice since there was limited mortality relative topVD1-WT vaccinated mice. Serum specimens were collected and DENV-2 Nt₅₀,viremia, and ES-ELISA titers were determined. DENV-2 neutralization wasstrongly correlated with survival; pVD1-CRR immunized mice exhibited arapid, large magnitude rise in DENV-2 neutralization whereas in pVD1-WTimmunized mice, neutralization was lower and increased more slowly(p<0.0001 in a 2-way ANOVA, FIG. 3E). 3 DPC Nt₅₀ titers of pVD1-CRRvaccinated mice were nearly 100 times greater than those of pVD1-WTvaccinated mice (CRR=2783.6, WT=29.5; Bonferroni posttest p<0.001). Thiswas a greater than 100-fold increase in DENV-2 neutralization comparedto pre-challenge for CRR vaccinated mice whereas it was a two-folddecrease over the same time period for WT vaccinated mice. Nt₅₀ titersof CRR vaccinated mice remained high 4 and 5 DPC whereas those of WTvaccinated mice slowly increased and remained 2-fold lower than CRRvaccinated mice by 5 DPC. Viremia was negatively correlated with DENV-2neutralization and positively correlated with mortality (FIG. 3F). 3 DPCmean viremia of WT vaccinated mice was 33 times higher (2325 FFU/ml)than that of CRR vaccinated mice (70 FFU/ml; Bonferroni posttestp<0.001) with two of four CRR immunized mouse sera below the limits ofassay detection (10 FFU/ml). WT viremia continued to increase 4-5 DPCand by 5 DPC was at least 1800 times higher than for CRR immunized micewhere all individuals had dropped to below detectable levels(ave=1.8×10⁴ and <10 FFU/ml for WT and CRR immunized mice respectively;p<0.001 with Bonferroni posttest in 2-way ANOVA). These findings implythat even though some CRR vaccinated mice were terminally ill 5 DPC,they had cleared their viremia, whereas WT vaccinated mice stillexhibited increasing viremia during this time of maximal mortality.Thus, the rapid, large magnitude increase in DENV-2 neutralization ofpVD1-CRR vaccinated mice was consistent with an anamnestic response orpotentially redirected immunity, whereas the slow rise in DENV-2neutralization of WT vaccinated mice was more characteristic of aprimary immune response.

pVD1-WT vaccinated AG129 mice suffering from lethally enhanced DENV-2disease exhibited pathology consistent with ADE disease described inpassive transfer studies with this mouse model (Balsitis et al., PLoSPathog 6:e1000790, 2010; Zellweger et al., Cell Host Microbe 7:128-139,2010). 3 DPC neither vaccinated nor naïve mice exhibited visiblepathological symptoms upon necropsy. All terminally ill mice however hadpale, blood-depleted livers, increased intestinal capillary blood flow,and enlarged gaseous stomachs, symptomatic of fluid accumulation causedby vascular leakage. Only WT vaccinated mice exhibited the most severegastrointestinal hemorrhage (FIG. 4A). Although gross pathology uponnecropsy of vaccinated mice was not observed until 4 DPC, hematoxylinand eosin staining of liver tissue revealed hepatitis pathology 3 DPCwith increasing severity 4 and 5 DPC. Consistent with the reduced grossmorbidity and mortality of pVD1-CRR compared to pVD1-WT vaccinated mice,CRR vaccinated mice reaching terminal endpoints exhibited mildlymphoplasmacytic portal, multifocal suppurative, and necrotizinghepatitis, including vacuolar vein congestion. However, pVD1-WTvaccinated mice exhibited moderate to severe lymphoplasmacytic,necrotizing, and multifocal portal hepatitis and in some individualsextensive vascular thrombosis and advanced vacuolar degeneration (FIG.4B).

Severe DENV pathology via ADE posits that there should be increasedinfection of FcγR bearing monocytic cells in humans (Morens, Clin InfectDis 19:500-512, 1994) and in AG129 mice, previous studies found thatantibody-enhanced mortality was associated with increased DENV infectionand replication in liver sinusoidal endothelial cells (Balsitis et al.,PLoS Pathog 6:e1000790, 2010; Zellweger et al., Cell Host Microbe7:128-139, 2010). Immunostaining for DENV-2 NS1 protein, which isexpressed during viral replication, was used to assess viral replicationin liver tissue. Of note, although no attempt was made to quantifydifferences between WT and CRR vaccine treatments, mononuclearinflammatory cells in the portal areas and sinusoidal endothelial cellsof both WT- and CRR-vaccinated liver tissue were NS1 positive by 3 DPC,suggestive of active viral replication in FcγR bearing cells of theliver, and liver tissue of naïve mice was negative for NS1 byimmunostaining (FIG. 4B). Lastly, pre-challenge pVD1-WT vaccinated mouseserum (1 day prior to DENV-2 challenge) significantly enhanced DENV-2replication in FcγR bearing human K562 cells, and pVD1-CRR immunizedmouse sera did not (FIG. 4C).

In summary, the high levels of DENV-1 vaccine induced cross-reactiveEDII_(FP) IgG prior to challenge, increased pathology and mortality,higher viremia, and active viral replication in FcγR bearing cells of WTimmunized mice are all consistent with previous descriptions ofantibody-enhanced DENV disease in AG129 mice (Balsitis et al., PLoSPathog 6:e1000790, 2010; Zellweger et al., Cell Host Microbe 7:128-139,2010).

Cross-Reactivity Reduced DNA Vaccination Sculpts Immune Memory so thatSubsequent Response to Virus Infection is Redirected, ReducingPathological and Increasing Protective Immunity

DENV-1 and DENV-2 epitope-specific ELISA was utilized to quantify anddifferentiate humoral immune responses to DENV-1 vaccination from DENV-2challenge. Supporting the hypothesis that WT vaccinated mice were slowerto mount DENV-2 specific immunity due to DENV-1 primed cross-reactivememory responses, WT vaccinated mice had lower DENV-2 total IgG titersyet larger populations of both DENV-1 and DENV-2 EDII_(FP) IgG than didCRR vaccinated mice following heterologous challenge (FIG. 5). TotalDENV-1 IgG titers did not differ between vaccine treatments 3, 4, and 5DPC with DENV-2 (FIG. 5A); however, DENV-2 IgG titers were higher forpVD1-CRR than for pVD1-WT immunized mice (FIG. 5B). Nevertheless,pVD1-WT vaccinated mice had significantly larger populations ofEDII_(FP) IgG for both DENV-1 (FIG. 5C) and DENV-2 (FIG. 5D) than didpVD1-CRR vaccinated mice. Not only did pVD1-WT mice have largerpopulations of cross-reactive EDII_(FP) IgG following heterologouschallenge, there was also a significant increase in DENV-1 EDII_(FP) IgGpost challenge when compared to the 12 week pre-challenge data (FIG.5C). DENV-1 EDII_(FP) IgG GMT of WT immunized mice increased from17.7±4.6% pre-challenge to 35.5±5.4% of the total IgG response postDENV-2 challenge. However, there was no increase in DENV-1 EDII_(FP) IgGfor CRR immunized mice before and after challenge (p=0.364, Mann-WhitneyU). Individual pairs of pre- and post-DENV-2 challenge sera from WTimmunized mice were also analyzed for DENV-1 EDII_(FP) IgG and thisanalysis also demonstrated a significant increase post-challenge(p=0.0138) and a significant effect of individual serum pairing(p=0.0267, one-tailed paired t-test). Together, these resultsdemonstrate a strong memory response to cross-reactive E proteinepitopes in DENV-2 that were primed by pVD1-WT vaccination and that suchan immunodominant anamnestic response was lacking in pVD1-CRR immunizedmice.

ES-ELISA can also determine the proportion of IgG recognizing E epitopesoutside of the manipulated EDII_(FP) and EDIII_(CR) antigenic regions inthe pVD1-CRR vaccine (non-EDII_(FP)EDIII_(CR) IgG; FIG. 1)(Hughes etal., Virol Journal 9:115, 2012). It was hypothesized that pVD1-CRRimmunized mice, with their reduced EDII_(FP) response, would exhibit anincreased capacity to redirect humoral immune responses to increase theDENV-2 non-EDII_(FP)EDIII_(CR) antibody populations concomitant withtheir reduced EDII_(FP) response and that WT immunized mice would showthe opposite pattern. Supporting this prediction, CRR vaccinated micehad significantly larger populations of DENV-2 non-EDII_(FP)EDIII_(CR)IgG 3, 4, and 5 DPC with DENV-2 than did pVD1-WT immunized mice(p=0.0025 in 2-way ANOVA; FIG. 5E). This was the same time period thatWT immunized mice exhibited maximum enhanced DENV-2 disease mortality(FIG. 3D) and significant increases in EDII_(FP) IgG (FIGS. 5C and 5D);whereas CRR immunized mice exhibited 100-fold increased DENV-2neutralization (FIG. 3E). Moreover, a comparison of DENV-1non-EDII_(FP)EDIII_(CR) IgG for individual WT immunized mice pre- andpost-challenge showed a significant decrease (p=0.0435) in this antibodypopulation 3-5 DPC and a significant effect of individual mouse serumpairing on this decrease (p=0.0111, one-tailed paired t-test). Adecrease in this DENV-1 antibody population of WT immunized mice is whatone would expect if there is a trade-off between immunodominantsecondary responses to EDII_(FP) and non-EDII_(FP)EDIII_(CR) epitopes.Antibody stimulated from epitopes outside EDII_(FP) and EDIII_(CR)antigenic regions altered in the pVD1-CRR vaccine include neutralizingDENV complex reactive antibodies such as MAb 1B7 primed by eithervaccine and boosted by DENV-2 challenge (Table 2) and/or DENV-2serotype-specific neutralizing antibodies stimulated only by thechallenge virus.

Three non-mutually exclusive mechanisms could explain the rapid increasein DENV-2 neutralizing antibody by CRR immunized mice 3 DPC: 1)anamnestically increased complex cross-reactive neutralizing antibodynot altered by the substitutions introduced in the pVD1-CRR vaccine(e.g., 1B7-like), 2) primarily increased DENV-2 specific neutralizingantibody, or 3) increased relative neutralizing capability of CRRvaccinated sera by these or other neutralizing antibody populations dueto a lack of steric interference by the large populations of EDII_(FP)IgG present in WT vaccinated mice (Ndifon et al., Proc Natl Acad Sci USA106:8701-8706, 2009). To test if the increased Nt₅₀ titers of CRRvaccinated mice were due to increases in DENV complex cross-reactiveneutralizing antibodies, the Nt₅₀ titers for CRR and WT vaccinated mouseserum 3 DPC against DENV-1, DENV-3, and DENV-4 were determined (FIG.5F). The mean (n=4 each) Nt₅₀ titers for CRR and WT vaccinated micerespectively were 46 and 33 for DENV-1 (p=0.066), 55 and 27 for DENV-3(p=0.0009), and 147 and 42 for DENV-4 (p=0.0178; one-tailed t-test forall). These 1.5-, 2.0- and 3.5-fold higher DENV-1, -3, and -4 Nt₅₀titers for CRR vaccinated mice did not approach the nearly 100-foldgreater DENV-2 Nt₅₀ titer of CRR immunized mice 3 DPC (2785 and 29respectively). These data indicate that CRR vaccinated mice haveincreased complex cross-reactive neutralizing antibody relative to WTvaccinated mice; but this antibody class either neutralizes DENV-2 moreefficiently than other DENV serotypes, or there are also increasedproportions of DENV-2 specific neutralizing antibody in CRR vaccinatedserum by 3 DPC with DENV-2.

To further characterize the epitopes recognized by the antibodiesresponsible for the rapid rise in DENV-2 neutralization observed in CRRvaccinated mice, the ability of 3 DPC sera to block the binding oflabeled MAbs was tested in a DENV-2 epitope-blocking ELISA. The blockingby WT and CRR vaccinated mouse sera for MAbs recognizing complexcross-reactive epitopes present in both DENV-1 vaccines and in theDENV-2 challenge virus (1B7, 10-D35A, and D3-5C9-1), MAbs recognizingsub-complex cross-reactive epitopes knocked out in the CRR vaccine butpresent in the WT vaccine and DENV-2 challenge virus (1A1D-2 and 9D12),and MAbs recognizing DENV-2 serotype-specific epitopes present only inthe challenge virus (9A3D-8 and 3H5) was examined. pVD 1-CRR vaccinatedmouse sera exhibited significantly greater blocking than did WTvaccinated sera for all of these MAbs with the exception of D3-5C9-1 theonly non-neutralizing MAb against DENV-2, indicating that CRR immunizedmice had higher titers of antibody recognizing the same or similarepitopes as these labeled MAbs (FIG. 6). The greatest relative increasein blocking was for DENV complex, neutralizing MAb 1B7 which exhibited16-fold greater blocking by CRR than the 2.25% blocking by WT (p=0.0072,two-tailed t-test), indicating greater 1B7-like neutralizing antibody inCRR vaccinated mouse sera than in WT mouse sera, and consistent withincreased anamnestic induction of normally sub-dominant 1B7-likeantibody in CRR immunized mice. CRR vaccinated mice also had 3.7 and1.6-fold larger populations of DENV-2 serotype-specific potentlyneutralizing 3H5 and 9A3D-8 like antibodies than did WT vaccinated mice(p=0.0003 and p=0.0058 respectively), 2.4 and 2.0-fold greatersub-complex cross-reactive neutralizing 1A1D-2 and 9D12 like antibodies(p=0.0001 and p<0.0001 respectively), and 3.1-fold more DENV complexcross-reactive weakly neutralizing 10-D35A like antibodies (p=0.0026).There was a non-significant 2.1-fold increase in complex cross-reactiveMAb D3-5C9-1.

Next, the neutralizing capabilities of these seven MAbs were determinedsince not all had been previously published in the literature (FIG. 6).DENV-2 serotype specific MAbs 3H5 and 9A3D-8 exhibited potent DENV-2neutralization (NT₅₀=0.15 and 0.94 μg/mL respectively). 1A1D-2 alsopotently neutralized DENV-2 (0.46 μg/mL) although it neutralized DENV-1and DENV-3 about 3-fold less than DENV-2, similar to the DENV-2neutralizing capabilities of both 9D12 and 1B7. 10-D35A exhibited weakneutralization of DENV-2 (13.04 μg/mL), and D3-5C9-1 did not neutralizeany DENV serotype. Together these results begin to tease apart theindividual humoral immune components responsible for the polyclonalresponse of vaccinated mice to heterologous infection. The 100-foldincrease in DENV-2 neutralization appeared to be due to a combination oflarge increases in cross-reactive neutralizing antibodies, similar to1B7, but also to relative increases and large populations of potentlyneutralizing serotype-specific and sub-complex cross-reactiveantibodies. Thus, in response to heterologous DENV-2 infection, pVD1-CRRimmunized mice exhibited rapid increases in neutralizing antibodypopulations with diverse patterns of reactivity that not only increasedprotection from enhanced DENV-2 disease mortality but also exhibitedincreased neutralization breadth across the DENV complex.

To further define the epitopes recognized by the neutralizing antibodiesresponsible for the rapid rise in DENV-2 neutralization observed in CRRvaccinated mice, the ability of 3 DPC sera to block the binding oflabeled MAbs was tested in a DENV-2 epitope-blocking ELISA. The blockingby WT and CRR vaccinated mouse sera was examined for MAbs recognizingcomplex cross-reactive epitopes present in both DENV-1 vaccines and inthe DENV-2 challenge virus (1B7, 10-D35A, and D3-5C9-1), MAbsrecognizing sub-complex cross-reactive epitopes knocked out in the CRRvaccine but present in the WT vaccine and DENV-2 challenge virus (1A1D-2and 9D12), and MAbs recognizing DENV-2 serotype-specific epitopespresent only in the challenge virus (9A3D-8 and 3H5). Three DPC pVD1-CRRvaccinated mouse sera exhibited significantly greater blocking than didWT vaccinated sera for all of these classes of MAbs, implying that CRRvaccinated sera had more antibody recognizing the same or similarepitopes as the labeled MAbs than did WT vaccinated sera (Table 3).

To standardize the percent blocking of these labeled MAbs by vaccinatedmouse sera and facilitate comparison between antibodies, the folddifference in average MAb blocking by CRR vaccinated mouse sera comparedto that of WT vaccinated mouse sera was determined for each MAb. Thegreatest increase in blocking by CRR sera compared to WT sera was forDENV complex, neutralizing MAb 1B7 which was 16-fold greater for CRRvaccinated than for WT vaccinated mouse sera (p=0.0006, two-tailedt-test comparing WT and CRR percent blocking values). This resultimplies that there was much greater 1B7 like neutralizing antibody inCRR vaccinated mouse sera than in WT mouse sera 3 DPC, and is consistentwith increased anamnestic induction of 1B7 like antibody in CRR relativeto WT vaccinated mice. Following similar logic, CRR vaccinated mice had3.7 and 1.6-fold larger populations of DENV-2 serotype-specific potentlyneutralizing 9A3D-8 and 3H5 like antibodies than did WT vaccinated mice,2.4 and 2.0-fold greater sub-complex cross-reactive neutralizing 1A1D-2and 9D12 like antibodies, and 3.1 and 2.1-fold more DENV complexcross-reactive weakly and non-neutralizing 10-D35A and D3-5C9-1 likeantibodies respectively. Lastly, the neutralizing capabilities of theseseven MAbs were determined since not all had been previously publishedin the literature (Table 3). DENV-2 serotype specific MAbs 3H5 and9A3D-8 showed the strongest DENV-2 neutralization (NT₅₀=0.15 and 0.94μg/mL respectively). 1A1D-2 also potently neutralized DENV-2 (0.46μg/mL) although it neutralized DENV-1 and DENV-3 about 3 times less thanDENV-2 which was similar to the moderate DENV-2 neutralizingcapabilities of both 9D12 and 1B7. D3-5C9-1 or 10-D35A did notneutralize any serotype strongly but 10-D35A did exhibit weakneutralization of DENV-2 (13.04 μg/mL). These results support thepattern of neutralization observed with polyclonal vaccinated mouseserum 3 DPC, suggesting that the 100-fold greater DENV-2 neutralizingcapability of CRR vaccinated mouse serum was due mostly to increasedpopulations of potently neutralizing DENV-2 type-specific and somesub-complex cross-reactive antibodies, and less so from increasedpopulations of moderately neutralizing, cross-reactive antibodies like1B7 and 9D12.

Thus, the greater than 100-fold increase in DENV-2 neutralization bypVD1-CRR vaccinated mice 3 DPC with DENV-2 was due to rapid, largeincreases in neutralizing antibody populations with diverse patterns ofcross-reactivity that not only effectively protected mice from enhancedDENV-2 disease mortality but also exhibited significantly increasedneutralization breadth across the DENV complex. This pattern ofincreased breadth of cross-neutralization by CRR vaccination bodes wellfor the use of tetravalent dengue CRR vaccines to rapidly inducebalanced and protective tetravalent immunity, a dengue vaccine goal thatcontinues to elude vaccine developers.

Taken together, these findings demonstrate that pVD1-WT vaccinated miceexhibited a classic pathological pattern of ‘original antigenic sin’with anamnestic immune responses to immunodominant cross-reactiveepitopes stimulated by the vaccine that not only interfered withsubsequent DENV-2 specific immunity but produced increasedimmunopathology. Conversely, pVD1-CRR vaccinated mice were not burdenedby original antigenic sin and were able to capitalize on it to rapidlymount a large magnitude, redirected, neutralizing immune response,effective at controlling DENV-2 viremia that was lacking in WTvaccinated mice.

TABLE 1 Nucleotide sequences of mutagenic primersPrimer Sequence (5′-3′)¹ SEQ ID Primer Nucleotide SubstitutionAmino Acid Substitution NO: D1-G106RTTCCTTTCCGAAGAGACGACAGCCATTGCCCCAGCC 13 GGG-CGT Gly-Arg D1-L107DTTCCTTTTCCGAAATCCCCACAGCCATTGCCCCAG 14 CTC-GAT Leu-Asp D1-G106RL107DCTTCCTTTTCCGAAATCCCGACAGCCATTGCCCCAGCC 15 GGA-CGG Gly-Arg CTC-GATLeu-Asp D1-K310D AGCCACTTCGTCCTCTAGCTTGAATGAGCCTGTGC 16 AAA-GAC Lys-AspD1-E311K TCAGCCACCTTTTTCTCTAGCTTGAATGAGCCTGTGC 17 GAA-AAG Glu-LysD1-K310DE311K GGGTCTCAGCCACCTTGTCCTCTAGCTTGAATGAGCCTGTGC 18 AAA-GACLys-Asp GAA-AAG Glu-Lys D1-P364QGCCTCAATGTTGACCTGTTTTTCTTTGTCAGTGACTATGGG 19 CCA-CAG Pro-Gln ¹Mutatednucleotides are shown in bold

TABLE 2 MAb reactivities for DENV-1 VLP mutants¹ MAb: MHAF 4G2 6B6C-14A1B-9 23-1 23-2 5H3 5-2 CR²: polyclonal group group group group groupgroup sub grp. Virus³: VLP construct % secrt'n D1 D2 SLEV MVEV WNV JEVYFV JEV WT DENV-1⁴ 100 5.7 6.6 5.1 4.8 5.4 6.6 5.1 4.5 G106R 200 100<0.1 12.5 1.5 25 100  nd⁵ 100 L107D 150 100 <0.1 50 100 <0.1 25 nd <0.6G106RL107D 130 100 <0.1 0.8 1.5 <0.1 <0.1 0.8 3.0 K310D 63 100 <0.1 100100 100 100 nd 150 E311K 50 100 100 100 100 100 100 nd 50 P364Q 50 100<0.1 100 100 100 100 nd 50 K310DE311K 10 100 100 50 12.5 100 100 nd 50P364Q G106RL107D 91 100 <0.1 3.0 <0.1 <0.1 <0.1 <0.1  <0.6 K310DE311KP364Q MAb: D3- 10- 20-783- MDVP- D2- 1B7 5C9-1 D35A 74014 1A1D-2 9D1255A 1F1-3 CR²: sub sub sub sub type- grp. comp comp comp comp comp compspec. Virus³: VLP construct D2 D4 DENV DENV D2 D1 DENV D1 WT DENV-1⁴ 6.34.7 5.7 5.1 5.7 5.1 4.2 6.3 G106R 0.8 100 nd nd 25 100 nd 100 L107D 50100 nd nd 100 100 nd 100 G106RL107D 100 100 nd nd 100 50 nd 100 K310D100 100 100 <0.1 1.5 <0.8 <1.3 100 E311K 100 100 100 3.9 50 <0.16 5.6100 P364Q 100 100 100 50 100 100 100 100 K310DE311K 100 100 100 <0.1<0.8 <0.16 <0.6 100 P364Q G106RL107D 100 100 50 <0.1 <0.1 <0.1 <0.6 100K310DE311K P364Q ¹Reactivity levels for MAbs exhibiting varyingcross-reactivity (CR) selected from different flaviviruses for wild-type(WT) and mutant VLP. ²MHAF is polyclonal murine hyper-immune asciticfluid, group CR antibodies recognize all viruses of at least the fourmajor pathogenic flavivirus serocomplexes; sub-group CR MAbs recognizeall or some members of two or more different flavivirus serocomplexes(e.g., MAb 5-2 recognizes JEV, DENV-1 and DENV-2 respectively); complex(comp) and sub-comp CR MAbs recognize all four DENV complex viruses or asubset thereof respectively, and type-specific MAbs recognize onlyDENV-1. ³Virus the MAb was raised against; D1 = dengue virus serotype 1(DENV-1), D2 = DENV-2, D3 = DENV-3, D4 = DENV-4, SLEV = St. Louisencephalitis virus, MVEV = Murray Valley encephalitis virus, WNV = WestNile virus, JEV = Japanese encephalitis virus, YFV = yellow fever virus.MAbs 20-783 and MDVP-55A are commercial MAbs raised against ‘denguevirus’. ⁴MAb reactivities for wild-type (WT) DENV-1 VLP are presented asinverse log₁₀ Ag-capture ELISA endpoint values and all mutant VLPs aspercent of remaining reactivity compared to WT. Emboldened valuesrepresent reactivity reductions greater than 90% relative to WT. ⁵nddenotes not determined.

TABLE 3 pVD1-CRR vaccine increases the immunodominance of diverseneutralizing antibody populations following heterologous DENV-2challenge MAb: 1B7 9A3D-8 3H5 1A1D-2 9D12 10-D35A D3-5C9-1 Crossreactivity comp+ type type subcomp. subcomp. complex complex Percentblocking 37.0 78.6 66.6 66.2 62.7 55.7 121.5 by pVD1-CRR vaccinatedsera¹ Percent blocking 2.3 21.2 40.9 27.3 31.8 18.2 59.1 by pVD1-WTvaccinated sera Fold pVD1-CRR 16.4 3.71 1.63 2.43 1.97 3.06 2.06increase in antibody population relative to pVD1-WT² p-value³ 0.0006<0.0001 0.0001 <0.0001 0.0001 0.0014 0.0289 Nt₅₀ (μg/mL) DENV-2 1.620.94 0.15 0.46 1.96 13.04 >100 DENV-1 0.32 >20 >20 1.68 1.21 64.18 >100DENV-3 2.12  nd⁴ nd 1.26 1.64 >100 >100 DENV-4 3.0 ndnd >20 >20 >100 >100 This table presents the results from anepitope-blocking ELISA conducted on pVD1-WT and pVD1-CRR vaccinatedAG129 mouse sera three days post challenge (DPC) with a sub-lethal doseof DENV-2. ¹Average percent blocking of labeled MAbs by vaccinated mousesera from four independent epitope-blocking ELISA assays each conductedon pools of 3 DPC sera from four pVD1-CRR vaccinated mice and fourpVD1-WT vaccinated mice. ²Fold increase in CRR vaccinated relative to WTvaccinated mouse sera for the blocking of labeled MAbs. Emboldenedvalues are significantly greater percent blocking by CRR vaccinatedsera. ³p-values from unpaired two-tailed t-test comparing percentblocking of labeled MAbs by pooled CRR vaccinated and WT vaccinatedmouse serum. ⁴nd = not determined

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. An isolated cross-reactivity reduced dengue virus E-glycoproteinpolypeptide, wherein the polypeptide is selected from: (1) a dengueserotype 1 virus (DENV-1) E-glycoprotein polypeptide comprising anarginine at position 106, an aspartic acid at position 107, an asparticacid at position 310, a lysine at position 311, a glutamine at position364, an isoleucine at position 468, a threonine at position 478, avaline at position 482 and a leucine at position 487, numbered withreference to SEQ ID NO: 1; (2) a dengue serotype 2 virus (DENV-2)E-glycoprotein polypeptide comprising an arginine at position 106, anaspartic acid at position 107, an glutamic acid at position 310, anarginine at position 311, an arginine at position 364, an isoleucine atposition 468, a threonine at position 478, a valine at position 482 anda leucine at position 487, numbered with reference to SEQ ID NO: 2; (3)a dengue serotype 3 virus (DENV-3) E-glycoprotein polypeptide comprisingan aspartic acid at position 106, an aspartic acid at position 107, anaspartic acid at position 308, a lysine at position 309, an asparticacid at position 362, an isoleucine at position 466, a threonine atposition 476, a valine at position 480 and a leucine at position 485,numbered with reference to SEQ ID NO: 3; and (4) a dengue serotype 4virus (DENV-4) E-glycoprotein polypeptide comprising a glutamic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a glutamine at position 311, a glutamine at position 389,an isoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487, numbered with reference toSEQ ID NO:
 4. 2. The polypeptide of claim 1, wherein the amino acidsequence of the polypeptide is at least 95% identical to SEQ ID NO: 1,and wherein the polypeptide comprises an arginine at position 106, anaspartic acid at position 107, an aspartic acid at position 310, alysine at position 311, a glutamine at position 364, an isoleucine atposition 468, a threonine at position 478, a valine at position 482 anda leucine at position 487 of SEQ ID NO:
 1. 3. (canceled)
 4. Thepolypeptide of claim 1, wherein the amino acid sequence of thepolypeptide is at least 95% identical to SEQ ID NO: 2, and wherein thepolypeptide comprises an arginine at position 106, an aspartic acid atposition 107, an glutamic acid at position 310, an arginine at position311, an arginine at position 364, an isoleucine at position 468, athreonine at position 478, a valine at position 482 and a leucine atposition 487 of SEQ ID NO:
 2. 5. (canceled)
 6. The polypeptide of claim1, wherein the amino acid sequence of the polypeptide is at least 95%identical to SEQ ID NO: 3, and wherein the polypeptide comprises anaspartic acid at position 106, an aspartic acid at position 107, anaspartic acid at position 308, a lysine at position 309, an asparticacid at position 362, an isoleucine at position 466, a threonine atposition 476, a valine at position 480 and a leucine at position 485 ofSEQ ID NO:
 3. 7. (canceled)
 8. The polypeptide of claim 1, wherein theamino acid sequence of the polypeptide is at least 95% identical to SEQID NO: 4, and wherein the polypeptide comprises a glutamic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 310, a glutamine at position 311, a glutamine at position 389,an isoleucine at position 468, a threonine at position 478, a valine atposition 482 and a leucine at position 487 of SEQ ID NO:
 4. 9. Thepolypeptide of claim 1, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ IDNO:
 4. 10. An isolated dengue virus-like particle (VLP) comprising thepolypeptide of claim
 1. 11. The VLP of claim 10, further comprising adengue virus prM protein.
 12. A recombinant nucleic acid moleculeencoding the polypeptide of claim
 1. 13. The recombinant nucleic acidmolecule of claim 12, comprising a nucleotide sequence at least 95%,identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:12.
 14. The recombinant nucleic acid molecule of claim 12, comprisingthe nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO:
 12. 15. A vector comprising the recombinant nucleic acidmolecule of claim
 12. 16. An isolated cell comprising the vector ofclaim
 15. 17. A composition comprising the polypeptide of claim 1, and apharmaceutically acceptable carrier.
 18. The composition of claim 17,further comprising an adjuvant.
 19. A tetravalent dengue virus vaccine,comprising a recombinant nucleic acid molecule encoding the DENV-1polypeptide of SEQ ID NO: 1, a recombinant nucleic acid moleculeencoding the DENV-2 polypeptide of SEQ ID NO: 2, a recombinant nucleicacid molecule encoding the DENV-3 polypeptide of SEQ ID NO: 3, and arecombinant nucleic acid molecule encoding the DENV-4 polypeptide of SEQID NO:
 4. 20. A method of eliciting an immune response in a subjectagainst dengue virus, comprising administering to the subject atherapeutically effective amount of the vaccine of claim 19, therebyeliciting an immune response in the subject against dengue virus. 21-22.(canceled)
 23. A composition comprising a DENV-1 E-glycoproteinpolypeptide, a DENV-2 E-glycoprotein polypeptide, a DENV-3E-glycoprotein polypeptide and a DENV-4 E-glycoprotein polypeptide; or arecombinant nucleic acid molecule encoding the DENV-1 E-glycoproteinpolypeptide, a recombinant nucleic acid molecule encoding the DENV-2E-glycoprotein polypeptide, a recombinant nucleic acid molecule encodingthe DENV-3 E-glycoprotein polypeptide and a recombinant nucleic acidmolecule encoding the DENV-4 E-glycoprotein polypeptide, wherein: (1)the DENV-1 E-glycoprotein polypeptide comprising an arginine at position106, an aspartic acid at position 107, an aspartic acid at position 310,a lysine at position 311, a glutamine at position 364, an isoleucine atposition 468, a threonine at position 478, a valine at position 482 anda leucine at position 487, numbered with reference to SEQ ID NO: 1; (2)the DENV-2 E-glycoprotein polypeptide comprising an arginine at position106, an aspartic acid at position 107, an glutamic acid at position 310,an arginine at position 311, an arginine at position 364, an isoleucineat position 468, a threonine at position 478, a valine at position 482and a leucine at position 487, numbered with reference to SEQ ID NO: 2;(3) the DENV-3 E-glycoprotein polypeptide comprising an aspartic acid atposition 106, an aspartic acid at position 107, an aspartic acid atposition 308, a lysine at position 309, an aspartic acid at position362, an isoleucine at position 466, a threonine at position 476, avaline at position 480 and a leucine at position 485, numbered withreference to SEQ ID NO: 3; and (4) the DENV-4 E-glycoprotein polypeptidecomprising a glutamic acid at position 106, an aspartic acid at position107, an aspartic acid at position 310, a glutamine at position 311, aglutamine at position 389, an isoleucine at position 468, a threonine atposition 478, a valine at position 482 and a leucine at position 487,numbered with reference to SEQ ID NO:
 4. 24. The composition of claim23, wherein: (1) the amino acid sequence of the DENV-1 polypeptide is atleast 95% identical to SEQ ID NO: 1, and wherein the polypeptidecomprises an arginine at position 106, an aspartic acid at position 107,an aspartic acid at position 310, a lysine at position 311, a glutamineat position 364, an isoleucine at position 468, a threonine at position478, a valine at position 482 and a leucine at position 487 of SEQ IDNO: 1; (2) the amino acid sequence of the DENV-2 polypeptide is at least95% identical to SEQ ID NO: 2, and wherein the polypeptide comprises anarginine at position 106, an aspartic acid at position 107, an glutamicacid at position 310, an arginine at position 311, an arginine atposition 364, an isoleucine at position 468, a threonine at position478, a valine at position 482 and a leucine at position 487 of SEQ IDNO: 2; (3) the amino acid sequence of the DENV-3 polypeptide is at least95% identical to SEQ ID NO: 3, and wherein the polypeptide comprises anaspartic acid at position 106, an aspartic acid at position 107, anaspartic acid at position 308, a lysine at position 309, an asparticacid at position 362, an isoleucine at position 466, a threonine atposition 476, a valine at position 480 and a leucine at position 485 ofSEQ ID NO: 3; and (4) the amino acid sequence of the DENV-4 polypeptideis at least 95% identical to SEQ ID NO: 4, and wherein the polypeptidecomprises a glutamic acid at position 106, an aspartic acid at position107, an aspartic acid at position 310, a glutamine at position 311, aglutamine at position 389, an isoleucine at position 468, a threonine atposition 478, a valine at position 482 and a leucine at position 487 ofSEQ ID NO:
 4. 25. The composition of claim 23, wherein the DENV-1polypeptide comprises the amino acid sequence of SEQ ID NO: 1, theDENV-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2,the DENV-3 polypeptide comprises the amino acid sequence of SEQ ID NO: 3and the DENV-4 polypeptide comprises the amino acid sequence of SEQ IDNO:
 4. 26. A method of eliciting an immune response in a subject againstdengue virus, comprising administering to the subject a therapeuticallyeffective amount of the composition of claim
 23. 27. The method of claim26, further comprising administering a protein boost vaccination.
 28. Amethod of eliciting an immune response in a subject against denguevirus, comprising administering to the subject a therapeuticallyeffective amount of the polypeptide of claim 1, thereby eliciting animmune response in the subject against dengue virus.